TW202304480A - Methods for tumor infiltrating lymphocyte (til) expansion related to cd39/cd69 selection and gene knockout in tils - Google Patents

Abstract

Provided herein are TILs that are (i) CD39 ^LO/CD69 ^LOand/or CD39/CD69 double negative, (ii) CD39/CD69 double knock-out, or (iii) the combination of (i) and (ii). In some embodiments, the subject TILs are produced by genetically manipulating a population of TILs that have been selected for (i) CD39 ^LO/CD69 ^LOand/or CD39/CD69 double negative, (ii) CD39/CD69 double knock-out, or (iii) the combination of (i) and (ii) expression ( e.g., a (i) CD39 ^LO/CD69 ^LOand/or CD39/CD69 double negative, (ii) CD39/CD69 double knock-out, or (iii) the combination of (i) and (ii) enriched TIL population). Also provided herein are expansion methods for producing such genetically modified TILs and methods of treatment using such TILs.

Description

A method for TIL amplification associated with CD39/CD69 selection and gene knockout in tumor infiltrating lymphocytes (TIL)

Cross References to Related Applications

This application asserts U.S. Provisional Patent Application Nos. 63/163,730, filed March 19, 2021; 63/255,657, filed October 14, 2021; and 63/280,536, filed November 17, 2021 of priority, each of which is incorporated herein by reference in its entirety for all purposes.

Treatment of bulky, refractory cancers using adoptive autologous transfer of tumor infiltrating lymphocytes (TILs) represents a powerful therapeutic option for patients with poor prognosis. Gattinoni et al., Nat. Rev. Immunol. 2006, 6, 383-393. TILs are dominated by T cells, and IL-2-based TIL expansion followed by "Rapid Expansion Process" (REP) has become the preferred method for TIL expansion due to its speed and efficiency. Dudley et al., "Science" 2002 , 298, 850-54; Dudley et al., " J. Clin. Oncol. " 2005, 23, 2346-57; Dudley et al., "Clinical Journal of Oncology 2008 , 26, 5233-39; Riddell et al., Science 1992, 257, 238-41; Dudley et al., J. Immunother. 2003 , 26, 332-42. Many approaches to improve the response of melanoma to TIL therapy and to extend TIL therapy to other tumor types have been explored with limited success and the field remains challenging. Goff et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley et al., J. Clin. Oncol. 2008 , 26, 5233-39; Rosenberg et al., Clinical Oncol. Oncology Research ( Clin. Cancer Res. 》 2011, 17, 4550-57. Combination studies of single immune checkpoint inhibitors have also been described, but other studies are ongoing and additional therapeutic approaches are needed (Kverneland et al. Target (Oncotarget), 2020 , 11(22), 2092-2105).

Furthermore, current TIL manufacturing and treatment processes are limited by length, cost, sterility issues, and other factors described herein, severely limiting the potential to treat patients refractory to other checkpoint inhibitor therapies. There is an urgent need to provide TIL manufacturing methods and therapies based on such methods that are suitable for treating patients with few or no viable treatment options. The present invention meets this need by providing a shortened manufacturing method for producing TILs.

The present invention provides improved and/or shortened processes and methods for the production of TILs to generate populations of therapeutic TILs with increased therapeutic efficacy in treating cancer with TILs undergoing CD39/CD69 as described herein Preselection, CD39/CD69 knockout, or a combination thereof.

Provided herein are TILs that are (i) CD39/CD69 double negative, (ii) CD39/CD69 double knockout (eg, genetically modified to silence or reduce CD39/CD69 expression), or (iii) (i) and Combinations of (ii). In some embodiments, TILs of the invention are produced by genetically manipulating a population of TILs selected for: (i) CD39/CD69 double negative, (ii) CD39/CD69 double knockout (e.g., genetically modified to silencing or reduced expression of CD39/CD69), or (iii) a combination of (i) and (ii). Also provided herein are expansion methods for producing such genetically modified TILs and therapeutic methods using such TILs.

The present invention provides methods for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining from the individual (b) from (a) In the first TIL population, select CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL to obtain enriched CD39/CD69 double negative TIL population; (c) enrich CD39/CD69 double negative TIL as the case may be The population is added to the closed system; (d) performing a first expansion by culturing the CD39/CD69 double negative TIL population enriched in IL-2 containing cell culture medium to generate a second TIL population, wherein the second TIL population is An expansion is performed in a closed vessel providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain a second TIL population, and wherein the transition from step (c) to step (d) (e) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen presenting cells (APCs), to generate a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain a third population of TILs, wherein the third population of TILs is a therapeutic TIL population, wherein the second expansion is optionally provided with a second gas-permeable surface (f) collecting the third TIL population obtained from step (e), wherein the transition from step (e) to step (f) is optionally performed without opening the system; (g) transferring the collected third TIL population from step (f) to an infusion bag, wherein The transfer from step (f) to (g) is carried out optionally without opening the system; (h) cryopreserving the infusion from step (g) comprising the collected third TIL population using a cryopreservation process (i) administering to the subject a therapeutically effective dose of the third population of TILs from the infusion bag in step (h); and (j) optionally at any time prior to administering step (i) the genetically modified enriched Collecting CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations and/or second TIL populations and/or third TIL populations so that the administered third TIL populations include genetically modified TILs, the Genetically modified TILs include genetic modifications that reduce the expression of CD39 and CD69.

The present invention provides methods for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining from the individual processing a tumor sample from an individual into multiple tumor fragments or processing a tumor sample obtained from an individual into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from an individual or patient; (b) optionally the The tumor fragments or the tumor lysates are added to the closed system; (c) a first expansion is performed by culturing the first TIL population in a cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, Optionally wherein the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine ( Perifosine), Oridonin, Herbacetin, Tehranolide, Isoliquiritigenin, Scutellarin and Honokiol to produce A second TIL population, which is a TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative, wherein the first amplification is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first The expansion is carried out for about 3-14 days to obtain a second TIL population, and wherein the transition from step (b) to step (c) is optionally performed without opening the system; (d) by using additional IL -2, OKT-3 and antigen-presenting cells (APC) supplement the cell culture medium of the second TIL population to carry out the second expansion to produce the third TIL population, wherein the second expansion is carried out for about 7-14 days to obtain the third TIL population The TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is (e) collecting the third population of TILs obtained from step (e), wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (f) to an infusion bag, wherein the transfer from steps (e) to (f) is optionally without opening the system Proceeding; (g) cryopreserving the infusion bag comprising the collected third TIL population from step (f) using a cryopreservation process; (h) administering to the individual a therapeutically effective dose of the infusion from step (g) Bag of the third TIL population; and (i) optionally at any time prior to administering step (h), genetically modifying the first population of TILs, the second population of TILs, and/or the third population of TILs such that the administered third population of TILs comprises the genetically modified Modified TILs, the genetically modified TILs comprise a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides methods for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining from the individual processing a tumor sample from an individual into multiple tumor fragments or processing a tumor sample obtained from an individual into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from an individual or patient; (b) optionally the The tumor fragments or the tumor lysates are added to the closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 to generate a second TIL population, wherein The first amplification is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-14 days to obtain the second TIL population, and wherein from step (b) to step The conversion of (c) is carried out optionally without opening the system; (d) by supplementing with additional IL-2, OKT-3, antigen presenting cell (APC) and protein kinase B (AKT) inhibitors Cell culture medium of the second TIL population for the second expansion, optionally wherein the AKT inhibitor is selected from the group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK -2206, BAY 1125976, perifosine, oridonin, nosytin, tenolid, isoliquiritigenin, luteinin and honokiol to generate a third TIL population, which is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population, wherein the second expansion is carried out for about 7-14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) collected from step (e ) the third TIL population obtained, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) the collected third TIL population from step (f) Three TIL populations are transferred to an infusion bag, wherein the transfer from steps (e) to (f) is optionally performed without opening the system; (g) cryopreservation of the inclusions from step (f) using a cryopreservation process an infusion bag of the collected third TIL population; (h) administering to the individual a therapeutically effective dose of the third TIL population from the infusion bag in step (g); and (i) optionally in the administering step ( h) at any time prior to genetically modifying the first TIL population, the second TIL population, and/or the third TIL population such that the administered third TIL population comprises genetically modified TILs comprising reduced Genetic modification of expression of CD39 and CD69.

The present invention provides methods for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining from the individual processing a tumor sample from an individual into multiple tumor fragments or processing a tumor sample obtained from an individual into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from an individual or patient; (b) optionally the The tumor fragments or the tumor lysates are added to the closed system; (c) a first expansion is performed by culturing the first TIL population in a cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, Optionally, wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonium A, gossinthin, tenolid, isoliquiritigenin, luteinin, and honokiol to generate a second population of TILs that are CD39 ^LO /CD69 ^LO enriched and/or CD39/CD69 double negative TILs A population, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain a second TIL population, and wherein from step (b) to step The conversion of (c) is carried out optionally without opening the system; (d) by supplementing the first line with additional IL-2, OKT-3, antigen presenting cell (APC) and protein kinase B (AKT) inhibitors. Cell culture medium of two TIL populations is used for the second expansion, wherein the AKT inhibitor is selected from the group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK- 2206, BAY 1125976, perifosine, oridonin, nossinin, tenolid, isoliquiritigenin, luteinin, and honokiol to generate a third TIL population that is enriched for CD39 ^LO / CD69 ^LO and/or CD39/CD69 double-negative TIL population, wherein the second expansion is carried out for about 7-14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) collected from step (e) The third TIL population obtained, wherein the transition from step (d) to step (e) is optionally carried out without opening the system; (f) the collected third TIL from step (f) The TIL population is transferred to an infusion bag, wherein from step (e) to The transfer of (f) is performed without opening the system as appropriate; (g) cryopreserving the infusion bag from step (f) containing the third collected TIL population using a cryopreservation process; The subject is administered a therapeutically effective dose of the third population of TILs from the infusion bag in step (g); and (i) optionally at any time prior to administering step (h) the first population of TILs, the second population of TILs population and/or a third population of TILs such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) by taking a tumor sample obtained from the individual Processing into multiple tumor fragments or processing a tumor sample obtained from an individual into a tumor digest to obtain a first TIL population derived from a tumor resected from an individual; (b) selecting CD39 ^LO from the first TIL population in (a) /CD69 ^LO and/or CD39/CD69 double negative TIL to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population; (c) Enrich CD39 ^LO /CD69 ^LO and/or The CD39/CD69 double negative TIL population is added to the closed system; (d) by culturing the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population enriched in IL-2 containing cell culture medium A first amplification to produce a second population of TILs, wherein the first amplification is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain a second TIL populations, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) by treating with additional IL-2, OKT-3 and antigen presenting cells ( APC) supplemented with the cell culture medium of the second TIL population to perform a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the second expansion is optionally carried out in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) is carried out optionally without opening the system; (f) collected from step (e) the third TIL population, wherein the transition from step (e) to step (f) is optionally performed without opening the system; (g) converting the collected third TIL population from step (f) Transfer to an infusion bag, wherein the transfer from steps (f) to (g) is carried out without opening the system as appropriate; (h) cryopreservation from step (g) containing the collected an infusion bag of the TIL population; (i) administering to the subject a therapeutically effective dose of a third TIL population from the infusion bag in step (h); and (j) optionally at any time prior to administering step (i) The genetic modification enriches the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population such that the administered third TIL population comprises genetically modified TILs comprising reduced CD39 and CD69 Genetic modification of expression.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) by taking a tumor sample obtained from the individual processing into tumor fragments or processing a tumor sample obtained from an individual into a tumor digest to obtain a first population of TILs derived from a tumor resected from an individual; (b) optionally dividing the tumor fragments or the tumor lysate Added to a closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from The group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, Nolide, isoliquiritigenin, chrysanthemum and honokiol to generate a second TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is based on The situation is carried out in a closed container providing a first gas-permeable surface area, wherein the first amplification is carried out for about 3-11 days to obtain a second TIL population, and wherein the transition from step (b) to step (c) is optionally at Performed without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen presenting cells (APCs) to generate a third TIL A population, wherein the second amplification is performed for about 7-11 days to obtain a third TIL population, wherein the second amplification is optionally performed in a closed container providing a second gas-permeable surface area, and wherein from step (c) to step The transformation of (d) is optionally carried out without opening the system; (e) collecting the third TIL population obtained from step (e), wherein the transformation from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (f) to an infusion bag, wherein the transfer from steps (e) to (f) is optionally performed at (g) cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h) administering to the individual a therapeutically effective dose of the TIL from step (g) ) of the third TIL population in the infusion bag; and (i) optionally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time prior to administering step (h) such that the administered A third TIL population therewith comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) by taking a tumor sample obtained from the individual processing into tumor fragments or processing a tumor sample obtained from an individual into a tumor digest to obtain a first population of TILs derived from a tumor resected from an individual; (b) optionally dividing the tumor fragments or the tumor lysate added to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is optionally at Provided in a closed container with a first gas-permeable surface area, wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally at Performed without opening the system; (d) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors Second expansion, optionally wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifol New, oridonin, gossin, tenoride, isoliquiritigenin, chrysanthemum, and honokiol to generate a third TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/ CD69 double negative TIL population, wherein the second expansion is performed for about 7-14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally provided with a second gas permeable surface (e) collecting the third TIL population obtained from step (e), wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (f) to an infusion bag, wherein The transfer from steps (e) to (f) is optionally performed without opening the system; (g) cryopreserving the infusion bag from step (f) containing the collected TIL population using a cryopreservation process; (h) administering to the individual a therapeutically effective dose of the third population of TILs from the infusion bag in step (g); and (i) optionally genetically modifying the first population of TILs at any time prior to administering step (h) , the second TIL population and/or the third TIL population, such that the administered third TIL population comprises genetically modified TILs comprising genes that reduce the expression of CD39 and CD69 grooming.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) by taking a tumor sample obtained from the individual processing into tumor fragments or processing a tumor sample obtained from an individual into a tumor digest to obtain a first population of TILs derived from a tumor resected from an individual; (b) optionally dividing the tumor fragments or the tumor lysate Added to a closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from The group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, Nolide, isoliquiritigenin, chrysanthemum and honokiol to generate a second TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is based on The situation is carried out in a closed container providing a first gas-permeable surface area, wherein the first amplification is carried out for about 3-11 days to obtain a second TIL population, and wherein the transition from step (b) to step (c) is optionally at (d) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors. Two expansions, optionally wherein the AKT inhibitor is selected from the group consisting of: paltaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine , Rubescensin A, Gossin, Tenolide, Isoliquiritigenin, Parcetrin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 Double negative TIL population, wherein the second expansion is performed for about 7-14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally provided in a second gas permeable surface area (e) collecting the third TIL population obtained from step (e), wherein The transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (f) to an infusion bag, wherein from The transfer of steps (e) to (f) depends on whether (g) cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h) administering to the individual a therapeutically effective dose of the TIL from step (g) ) of the third TIL population in the infusion bag; and (i) optionally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time prior to administering step (h) such that the administered A third TIL population therewith comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core Obtaining and/or receiving a first TIL population by biopsy, mini-biopsy, or other means used to obtain a sample from a cancer in the patient or individual containing a mixture of tumor and TIL cells, (b) from (a) For the first TIL population, select CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL to obtain a TIL population enriched in PD-1; (c) enrich CD39 ^LO /CD69 ^LO and/or CD39/CD69 as appropriate The double negative TIL population is added to the closed system; (d) a first expansion is performed by culturing the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population enriched in IL-2 containing cell culture medium to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain a second population of TILs, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) by supplementing with additional IL-2, OKT-3 and antigen presenting cells (APCs) The cell culture medium of the second TIL population is used to carry out the second expansion to produce the third TIL population, wherein the second expansion is carried out for about 7-11 days to obtain the third TIL population, wherein the second expansion is optionally provided in the second TIL population. (2) carried out in a closed container with a gas-permeable surface area, and wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) collecting the third gas obtained from step (e) TIL population, wherein the transition from step (e) to step (f) is optionally performed without opening the system; (g) transferring the collected third TIL population from step (f) to infusion bags, wherein the transfer from steps (e) to (f) is optionally performed without opening the system; (h) cryopreserving the TIL population from step (g) containing the collected TIL population using a cryopreservation process an infusion bag; (i) administering to the subject a therapeutically effective dose of a third population of TILs from the infusion bag in step (h); and (j) optionally genetically modified at any time prior to administering step (i) Enriching CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations and/or second TIL populations and/or third TIL populations so that the administered third TIL populations comprise genetically modified TILs, the Such genetically modified TILs include genetic modifications that reduce the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core Obtaining and/or receiving a first population of TILs by biopsy, mini-biopsy, or other means used to obtain a sample containing a mixture of tumor and TIL cells from a cancer in the patient or individual, (b) as the case may be, Fragments or these tumor lysates are added to the closed system; (c) a first expansion is performed by culturing the first TIL population in cell culture medium containing IL-2 and a protein kinase B (AKT) inhibitor, optionally Wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin , Gossin, Tenolide, Isoliquiritigenin, Pheretin and Honokiol to generate a second TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is optionally performed in a closed vessel providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain a second population of TILs, and wherein from step (b) to step (c ) transformation was performed optionally without opening the system; (d) a second expansion was performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen presenting cells (APCs). to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein The transition from step (c) to step (d) is optionally carried out without opening the system; (e) collecting the third TIL population obtained from step (d), wherein from step (d) to step ( The conversion of e) is carried out without opening the system as appropriate; (f) transferring the collected third TIL population from step (e) to an infusion bag, wherein steps (e) to (f) The transfer is optionally performed without opening the system; (g) cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h) administering treatment to the individual an effective dose of the third TIL population from the infusion bag in step (g); and (i) optionally genetically modifying the first TIL population, the second TIL population, and/or the second TIL population at any time prior to administering step (h); Three populations of TILs, such that the third population of TILs administered comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core Obtaining and/or receiving a first population of TILs by biopsy, mini-biopsy, or other means used to obtain a sample containing a mixture of tumor and TIL cells from a cancer in the patient or individual, (b) as the case may be, The fragments or the tumor lysates are added to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to generate a second TIL population, wherein the first TIL population An expansion is optionally performed in a closed vessel providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein from step (b) to step (c (d) by supplementing the second IL-2, OKT-3, antigen-presenting cell (APC) and protein kinase B (AKT) inhibitors with additional IL-2, OKT-3, antigen-presenting cell (APC) and protein kinase B (AKT) inhibitors. Cell culture medium of the TIL population for the second expansion, optionally wherein the AKT inhibitor is selected from the group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206 , BAY 1125976, Perifosine, Oridonin, Gossytin, Tenolide, Isoliquiritigenin, Phetogenin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO / CD69 ^LO and/or CD39/CD69 double-negative TIL population, wherein the second expansion is carried out for about 7-11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is based on The case is carried out in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is carried out optionally without opening the system; (e) collected from step (d) the third TIL population, wherein the transition from step (d) to step (e) is optionally carried out without opening the system; (f) the collected third TIL from step (e) The population is transferred to an infusion bag, wherein the transfer from steps (e) to (f) is performed without opening the system as appropriate; (g) cryopreservation of the containing collected from step (f) using a cryopreservation process (h) administering to the individual a therapeutically effective dose of a third TIL population from the infusion bag in step (g); and (i) optionally administering any TIL population prior to step (h) Temporally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population such that the administered third TIL population comprises genetically modified TILs comprising reduced CD39 and Genetic modification of CD69 expression.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core Obtaining and/or receiving a first population of TILs by biopsy, mini-biopsy, or other means used to obtain a sample containing a mixture of tumor and TIL cells from a cancer in the patient or individual, (b) as the case may be, Fragments or these tumor lysates are added to the closed system; (c) a first expansion is performed by culturing the first TIL population in cell culture medium containing IL-2 and a protein kinase B (AKT) inhibitor, optionally Wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin , Gossin, Tenolide, Isoliquiritigenin, Pheretin and Honokiol to generate a second TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is optionally performed in a closed vessel providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain a second population of TILs, and wherein from step (b) to step (c ) conversion is performed optionally without opening the system; (d) by supplementing the second TIL with additional IL-2, OKT-3, antigen presenting cell (APC) and protein kinase B (AKT) inhibitors The cell culture medium of the population is used for the second expansion, wherein the AKT inhibitor is selected from the group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Rubescensin, Gossin, Tenolide, Isoliquiritigenin, Parifaxin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the second expansion is carried out for about 7-11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optional in a closed container providing a second air-permeable surface area, and wherein the transition from step (c) to step (d) is carried out optionally without opening the system; (e) collected from step (d) The third TIL population, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) the collected third TIL population from step (e) Transfer to infusion bag, wherein from step (e) to (f ) is transferred optionally without opening the system; (g) cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h) administering to the individual a therapeutically effective dose of the third TIL population from the infusion bag in step (g); and (i) optionally genetically modifying the first TIL population, the second TIL population, and/or at any time prior to administering step (h) A third population of TILs such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: resecting a tumor from the individual or patient, the the tumor comprises the first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into multiple tumor fragments; (c) enzymatically digesting the multiple tumor fragments to obtain a first TIL population; (d) selecting CD39 ^LO /CD69 ^LO and/or from the first TIL population in (c) Or CD39/CD69 double-negative TIL to obtain enriched CD39 ^LO / CD69 ^LO and/or CD39/CD69 double-negative TIL population; (e) Enrich CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative as appropriate The TIL population of is added to the closed system; (f) by culturing the TIL population enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative in a cell culture medium comprising IL-2 to carry out the first expansion, to generate a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain a second population of TILs, and wherein The transition from step (e) to step (f) is optionally performed without opening the system; (g) by supplementing the second cell with additional IL-2, OKT-3 and antigen presenting cells (APCs). The cell culture medium of the TIL population is used to perform the second expansion to produce the third TIL population, wherein the second expansion is carried out for about 7-11 days to obtain the third TIL population, wherein the second expansion is optionally provided with a second gas permeable carried out in a closed container of the surface area, and wherein the transition from step (f) to step (g) is optionally carried out without opening the system; (h) collecting the third population of TILs obtained from step (g), wherein the transition from step (g) to step (h) is optionally performed without opening the system; (i) transferring the collected third TIL population from step (h) to an infusion bag, wherein The transfer of (h) to (i) is performed without opening the system as appropriate; (j) cryopreserving the infusion bag containing the collected TIL population from step (i) using a cryopreservation process; (k) administering to the individual or patient with cancer a therapeutically effective dose of the third population of TILs from the infusion bag in step (g); and (l) optionally genetically modified at any time prior to administering step (k) Enriching CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population, second TIL population and/or third TIL population, so that the administered third TIL population includes Genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: resecting a tumor from the individual or patient, the the tumor comprises the first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into a plurality of tumor fragments; (c) enzymatically digesting the plurality of tumor fragments to obtain a first TIL population; (d) optionally adding tumor fragments or tumor digests to the closed system; (e) The first expansion is performed by culturing the first population of TILs in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: patasertide , GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin, Huangpi and honokiol to produce a second population of TILs that are CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplification is optionally provided before providing the first gas permeable surface area Performed in a closed vessel, wherein the first amplification is performed for about 3-11 days to obtain a second TIL population, and wherein the transition from step (d) to step (e) is optionally performed without opening the system; ( f) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (e) to step (f) is optionally performed in (g) collecting the third TIL population obtained from step (f), wherein the transition from step (f) to step (g) is optionally performed without an open system; ( h) transferring the collected third TIL population from step (g) to an infusion bag, wherein the transfer from steps (g) to (h) is optionally performed without opening the system; (i) using freezing Preservation cryopreserving the infusion bag comprising the collected TIL population from step (h); (l) administering a therapeutically effective dose of the infusion bag from step (h) to the individual or patient suffering from cancer the third TIL population; and (k) optionally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time prior to administering step (1) such that the administered third TIL population The L population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: resecting a tumor from the individual or patient, the the tumor comprises the first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into a plurality of tumor fragments; (c) enzymatically digesting the plurality of tumor fragments to obtain a first TIL population; (d) optionally adding tumor fragments or tumor digests to the closed system; (e) A first expansion is performed by culturing the first TIL population in cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is optionally in a closed vessel providing a first gas-permeable surface area wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein the transition from step (e) to step (f) is optionally performed without opening the system; (f) Second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors, optionally where AKT The inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, Gossin, tenolide, isoliquiritigenin, lutein and honokiol to generate the third TIL population, which is CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the third TIL population The second expansion is performed for about 7-11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein The transition from step (e) to step (f) is optionally carried out without opening the system; (g) collecting the third population of TILs obtained from step (f), wherein from step (f) to step (g ) is optionally performed without opening the system; (h) transferring the collected third TIL population from step (g) to an infusion bag, wherein the transfer from steps (g) to (h) is (i) cryopreserving the infusion bag containing the collected TIL population from step (h) using a cryopreservation process; (l) administering to the individual or patient with cancer and a therapeutically effective dose of the third TIL population from the infusion bag in step (h); and (k) optionally genetically modifying the first TIL population, the second TIL population at any time prior to administering step (l); The L population and/or the third population of TILs, such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: resecting a tumor from the individual or patient, the the tumor comprises the first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into a plurality of tumor fragments; (c) enzymatically digesting the plurality of tumor fragments to obtain a first TIL population; (d) optionally adding tumor fragments or tumor digests to the closed system; (e) The first expansion is performed by culturing the first population of TILs in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: patasertide , GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin, Huangpi and honokiol to produce a second population of TILs that are CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplification is optionally provided before providing the first gas permeable surface area Performed in a closed vessel, wherein the first amplification is performed for about 3-14 days to obtain a second TIL population, and wherein the transition from step (d) to step (e) is optionally performed without opening the system; ( f) Second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors, where AKT is inhibited as appropriate The agent is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, grass cotton flavin, tenolide, isoliquiritigenin, lutealin and honokiol to generate the third TIL population, which is CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population, in which the second The expansion is performed for about 7-11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein the The transition from step (e) to step (f) is optionally carried out without opening the system; (g) collecting the third population of TILs obtained from step (f), wherein from step (f) to step (g) The change system depends on the situation in the closed system (h) transferring the collected third TIL population from step (g) to an infusion bag, wherein the transfer from steps (g) to (h) is optionally without opening the system (i) cryopreserving the infusion bag comprising the collected TIL population from step (h) using a cryopreservation process; (l) administering a therapeutically effective dose of the TIL from step (h) to the individual or patient suffering from cancer ) of the third TIL population of the infusion bag; and (k) optionally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time prior to administering step (1) such that all The third population of TILs administered comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core Obtaining and/or receiving a first TIL population by biopsy, mini-biopsy, or other means for obtaining a sample from the individual or patient containing a mixture of tumor and TIL cells; (b) from the first TIL in (a) Population selection of CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population; (c) enrich CD39 ^LO /CD69 ^LO and /Or the CD39/CD69 double negative TIL population is contacted with the first cell medium; (d) carries out the initial expansion of the TIL population enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative in the first cell culture medium ( or initiate the first amplification) to obtain a second TIL population, wherein the number of the second TIL population is at least 5 times greater than that of the first TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody) and optionally antigen-presenting cells (APC), wherein the first expansion is initiated for a period of 1 to 8 days; (e) rapid second expansion of the second TIL population in a second cell culture medium increasing to obtain a third TIL population, wherein the third TIL population is at least 50-fold greater in number than the second TIL population after 7 to 8 days from the start of the rapid expansion; wherein the second cell culture medium comprises IL-2 , OKT-3 (anti-CD3 antibody), and APC; and wherein the rapid expansion is performed for a period of 14 days or less, and the rapid second expansion may be performed 1 day, 2 days after initiation of the rapid second expansion as appropriate , 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days; (f) collecting the third TIL population; (g) administering treatment to the individual or patient with cancer an effective portion of the third TIL population; and (h) optionally genetically modified to enrich CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations and/or the third TIL population at any time prior to administering step (g) The second population of TILs and/or the third population of TILs, such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core Obtaining and/or receiving a first TIL population by biopsy, mini-biopsy, or other means for obtaining a sample from the individual or patient containing a mixture of tumor and TIL cells; The first TIL population was cultured in cell culture media of optionally OKT-3 (anti-CD3 antibody), optionally antigen-presenting cells (APCs) and protein kinase B (AKT) inhibitors for initial expansion (or initiation of the second TIL population). - Expansion), where the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifol, as the case may be New, oridonin, gossin, tenoride, isoliquiritigenin, chrysanthemum, and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/ A population of TILs that are double negative for CD69, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the initial first expansion is performed for about 1-8 days to obtain a second population of TILs, and wherein The transition from step (a) to step (b) is optionally performed without opening the system; (c) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and APCs; and wherein the rapid expansion is performed for a period of 14 days or less, optionally the rapid second expansion can be at The rapid second amplification is initiated 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days; (d) collecting a third population of TILs; ( e) administering to the individual or patient with cancer a therapeutically effective portion of the third population of TILs; and (f) optionally genetically modifying the first population of TILs, the second population of TILs at any time prior to administering step (e); population and/or a third population of TILs such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core obtaining and/or receiving a first TIL population by biopsy, mini-biopsy, or other means for obtaining a sample from the individual or patient containing a mixture of tumor and TIL cells; (b) performing the first TIL population in a first cell culture medium; Initial expansion of the TIL population (or initiation of the first expansion) to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally Antigen Presenting Cells (APCs), wherein the initial first expansion is performed for a period of 1 to 8 days; (c) performing a rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), APC, and protein kinase B (AKT) inhibitors, where appropriate, wherein the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363 , GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenoride, isoliquiritigenin, chrysanthemum and honokiol to produce A third TIL population, which is a population of TILs enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative, wherein the rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion can be at The rapid second amplification is initiated 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days; (d) collecting a third population of TILs; ( e) administering to the individual or patient with cancer a therapeutically effective portion of the third population of TILs; and (f) optionally genetically modifying the first population of TILs, the second population of TILs at any time prior to administering step (e); population and/or a third population of TILs such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core Obtaining and/or receiving a first TIL population by biopsy, mini-biopsy, or other means for obtaining a sample from the individual or patient containing a mixture of tumor and TIL cells; The first TIL population was cultured in cell culture media of optionally OKT-3 (anti-CD3 antibody), optionally antigen-presenting cells (APCs) and protein kinase B (AKT) inhibitors for initial expansion (or initiation of the second TIL population). - Expansion), where the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifol, as the case may be New, oridonin, gossin, tenoride, isoliquiritigenin, chrysanthemum, and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/ A population of TILs that are double negative for CD69, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the initial first expansion is performed for about 1-8 days to obtain a second population of TILs, and wherein The transition from step (a) to step (b) is optionally performed without opening the system; (c) performing a rapid second expansion in a second cell culture medium to obtain a third population of TILs; wherein the second cells The culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), APC, and protein kinase B (AKT) inhibitor, where the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183 , AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenolid, isoliquiritigenin, chrysanthemum and honokiol, To generate a third population of TILs that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion This rapid second expansion can be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second expansion; (d) collecting a third population of TILs (e) administering to the individual or patient with cancer a therapeutically effective portion of the third population of TILs; and (f) optionally genetically modifying the first population of TILs, the third population of TILs at any time prior to administering step (e). The second TIL population and/or the third TIL population such that the third TIL population administered The bodies comprise genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) resecting the cancer from the individual or patient a tumor, the tumor comprising the first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) Fragmentation of tumors into tumor fragments or processing of tumors into tumor digests; (c) Selection of CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs from the first TIL population of tumor fragments to obtain enriched CD39 ^LO / CD69 ^LO and/or CD39/CD69 double-negative TIL population; (c) contacting the tumor fragments with the first cell culture medium; (d) enriching CD39 ^LO /CD69 ^LO and/or the first cell culture medium or an initial expansion (or initiation of a first expansion) of a CD39/CD69 double negative TIL population to obtain a second TIL population, wherein the second TIL population is at least 5-fold greater in number than the first TIL population, wherein the first Cell culture medium comprising IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs), wherein the first expansion is initiated for a period of 1 to 8 days; (e) at A rapid second expansion of the second TIL population to obtain a third TIL population, wherein the third TIL population is numerically greater than the second TIL population 7 to 8 days after the start of the rapid expansion at least 50-fold; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and APC; and wherein the rapid expansion is performed for a period of 14 days or less, optionally rapid second expansion can be at performing 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second amplification; (f) collecting the third TIL population; ( g) administering a therapeutically effective portion of the third TIL population to the individual or patient with cancer; and (h) optionally genetically modifying CD39 ^LO /CD69 ^LO at any time prior to administering step (g) and and/or a CD39/CD69 double negative TIL population and/or a second TIL population and/or a third TIL population such that the administered third TIL population comprises genetically modified TILs comprising reduced Genetic modification of expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) resecting the cancer from the individual or patient a tumor, the tumor comprising the first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) fragmenting the tumor into tumor fragments or processing the tumor into a tumor digest; APC) and a protein kinase B (AKT) inhibitor in cell culture medium for initial expansion (or initiation of the first expansion) by culturing the first TIL population, where the AKT inhibitor is selected from the group consisting of: Pa Taserti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifoxin, Oridonin A, Gossin, Tenoride, Isolicorice Magnolin, lutein and honokiol to generate a second population of TILs enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplification is optionally provided in the first gas permeable Performed in a closed vessel at the surface area, where the initial first amplification proceeds for about 1-8 days to obtain the second TIL population, and where the transition from step (a) to step (b) is optionally done in a closed system (d) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibodies) and APCs; and wherein the rapid expansion is performed for a period of 14 days or less, optionally the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days after initiation of the rapid second amplification days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days; (e) collecting the third TIL population; (f) administering a therapeutically effective portion of the first TIL population to the individual or patient with cancer. three TIL populations; and (g) optionally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time prior to administering step (f), such that the administered third TIL population comprises Genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) resecting the cancer from the individual or patient a tumor, the tumor comprising the first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) fragmenting the tumor into tumor fragments or processing the tumor into tumor digests; (c) performing an initial expansion (or initiating the first expansion) of the first TIL population in the first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APC), wherein the first expansion is initiated for 1 to 8 days Period; (d) rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), APC and protein kinase B (AKT ) inhibitors, wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, as the case may be , Rubescensin A, Gossin, Tenolide, Isoliquiritigenin, Parcetrin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 Double negative TIL population, wherein rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion can be performed 1 day, 2 days, 3 days, 4 days after initiation of the rapid second expansion days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days; (e) collecting the third TIL population; (f) administering a therapeutically effective portion of the first TIL population to the individual or patient with cancer. three TIL populations; and (g) optionally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time prior to administering step (f), such that the administered third TIL population comprises Genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) resecting the cancer from the individual or patient a tumor, the tumor comprising the first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) fragmenting the tumor into tumor fragments or processing the tumor into a tumor digest; APC) and a protein kinase B (AKT) inhibitor in cell culture medium for initial expansion (or initiation of the first expansion) by culturing the first TIL population, where the AKT inhibitor is selected from the group consisting of: Pa Taserti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifoxin, Oridonin A, Gossin, Tenoride, Isolicorice Magnolin, lutein and honokiol to generate a second population of TILs enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplification is optionally provided in the first gas permeable Performed in a closed vessel at the surface area, where the initial first amplification proceeds for about 1-8 days to obtain the second TIL population, and where the transition from step (a) to step (b) is optionally done in a closed system (d) rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody), APC and protein kinase B (AKT) inhibitor, where the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perid, as the case may be Fuxin, Rubescensin A, Gossin, Tenoride, Isoliquiritigenin, Parcetrin and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39 / CD69 double negative TIL population, wherein the rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion can be performed 1 day, 2 days, 3 days after initiation of the rapid second expansion , 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days; (e) collecting the third TIL population; (f) administering a therapeutically effective moiety to the individual or patient with cancer the third TIL population; and (g) optionally after administering step (f) At any time prior to genetically modifying the first TIL population, the second TIL population, and/or the third TIL population such that the administered third TIL population comprises genetically modified TILs comprising reduced CD39 and Genetic modification of CD69 expression.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core Obtaining and/or receiving a first TIL population by biopsy, mini-biopsy, or other means used to obtain a sample from a cancer in the patient or individual containing a mixture of tumor and TIL cells, (b) from (a) The first TIL population selects CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations; (c) by including IL- 2. Culture enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations in the first cell culture medium of OKT-3 and antigen-presenting cells (APCs) to initiate the first expansion to generate the second A population of TILs, wherein the initial first expansion is carried out in a container comprising a first gas-permeable surface area, wherein the initial first expansion is performed for a first period of about 1 to 11 days to obtain a second TIL population, wherein the second The TIL population is greater in number than the first TIL population; (d) optionally restimulating the second TIL population with OKT-3; (e) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified The second population of TILs comprises genetic modifications that reduce the expression of CD39 and CD69 such that the second population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs; (f) by including IL-2, OKT-3 and Rapid second expansion by culturing the modified second TIL population in the second medium of APCs to produce a third TIL population, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population comprising genetic modifications that reduce the expression of CD39 and CD69, such that the third population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL; (g) collecting the a third population of TILs; and (h) administering to the individual or patient with cancer a therapeutically effective portion of the third population of TILs.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core Obtaining and/or receiving a first TIL population by biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer in the patient or individual, (b) by including IL- 2. Culture the first TIL population in the first cell culture medium of OKT-3, antigen-presenting cells (APC) and protein kinase B (AKT) inhibitors to initiate the first amplification, wherein the AKT inhibitors are selected from The group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, Nolide, isoliquiritigenin, luteinin and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population where the first amplification was initiated is performed in a vessel comprising a first gas-permeable surface area, wherein the first expansion is initiated for a first period of about 1 to 11 days to obtain a second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (c) optionally restimulating the second TIL population with OKT-3; (d) genetically modifying the second TIL population to generate a modified second TIL population, wherein the modified second TIL population comprises reduced CD39 and CD69 Expressed genetic modification such that the second population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs; (e) by culturing the modified TILs in a second medium comprising IL-2, OKT-3 and APC The second TIL population is subjected to a rapid second expansion to produce a third TIL population, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain a therapeutic TIL population, wherein the third TIL population is comprising A genetically modified therapeutic TIL population that reduces the expression of CD39 and CD69 such that a third population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs; (f) collecting the third TIL population; and (g) contributing to The individual or patient with cancer is administered a therapeutically effective portion of the third TIL population.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core Obtaining and/or receiving a first TIL population by biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer in the patient or individual, (b) by including IL- 2. Culture the first TIL population in the second cell culture medium of OKT-3 and antigen-presenting cells (APC) to perform the initial first expansion to produce the second TIL population, wherein the initial first expansion is in performed in a container comprising a first gas-permeable surface area, wherein the initial first amplification is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL the population; (c) optionally restimulating the second TIL population with OKT-3; (d) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises reduced CD39 and Genetic modification of CD69 expression such that the second population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs; (e) by including IL-2, OKT-3, APC and protein kinase B (AKT) Rapid secondary expansion by culturing a modified second population of TILs in a secondary medium for inhibitors, optionally wherein the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenolid, isoliquiritigenin, lutein and honokiol to produce the first Three TIL populations that are enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein a rapid second expansion is performed for a second period of about 14 days or less to obtain a therapeutic TIL population, wherein The third TIL population is a therapeutic TIL population comprising genetic modifications that reduce the expression of CD39 and CD69 such that the third population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs; (f) collecting the third TIL population and (g) administering to the individual or patient with cancer a therapeutically effective portion of the third population of TILs.

The present invention provides a method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) consisting of surgical excision, biopsy, core Obtaining and/or receiving a first TIL population by biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer in the patient or individual, (b) by including IL- 2. Culture the first TIL population in the first cell culture medium of OKT-3, antigen-presenting cells (APC) and protein kinase B (AKT) inhibitors to initiate the first amplification, wherein the AKT inhibitors are selected from The group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, Nolide, isoliquiritigenin, luteinin and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population where the first amplification was initiated is performed in a vessel comprising a first gas-permeable surface area, wherein the first expansion is initiated for a first period of about 1 to 11 days to obtain a second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (c) optionally restimulating the second TIL population with OKT-3; (d) genetically modifying the second TIL population to generate a modified second TIL population, wherein the modified second TIL population comprises reduced CD39 and CD69 Expressed genetic modification such that the second population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs; (e) by including IL-2, OKT-3, APC and protein kinase B (AKT) inhibitors Rapid second expansion by culturing a modified second population of TILs in a second culture medium, optionally wherein the AKT inhibitor is selected from the group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC- 0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Rubescensin, Ressin, Tenolid, Isoliquiritigenin, Paretin and Honokiol to generate the third TIL A population, which is a TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain a therapeutic TIL population, wherein the third The TIL population is a therapeutic TIL population comprising genetic modifications that reduce the expression of CD39 and CD69 such that a third population comprises CD39 ^LO /CD69 ^{L O} and/or CD39/CD69 double-negative TILs; (f) collecting the third TIL population; (g) administering a therapeutically effective portion of the third TIL population to the individual or patient suffering from cancer.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by dividing The obtained tumor sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual's cancer; (b) selecting CD39 ^LO /CD69 ^LO from the first TIL population in step (a) and/or CD39/CD69 double-negative TILs to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations; (c) by including IL-2, OKT-3 and antigen-presenting cells ( APC) culture enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations in the first cell culture medium to initiate the first expansion to generate a second TIL population in which the first expansion is initiated is carried out in a container comprising a first gas-permeable surface area, wherein the first expansion is initiated for a first period of about 1 to 7/8 days to obtain a second population of TILs, wherein the number of the second population of TILs is greater than that of the first TILs population; (d) performing a rapid second expansion by culturing the second population of TILs in a second medium comprising IL-2, OKT-3, and APCs to produce a third population of TILs, wherein the rapid second The number of APCs added in the expansion is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain a third population of TILs, wherein The third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container comprising a second gas-permeable surface area; (e) collecting the therapeutic TIL population obtained from step (d); (f) converting The collected TIL population from step (e) is transferred to an infusion bag; and (g) genetically modified CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs are optionally enriched at any time prior to collection step (e) The TIL population and/or the second TIL population and/or the third TIL population such that the therapeutic TIL population comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by dividing The obtained tumor sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from an individual's cancer; ) and a protein kinase B (AKT) inhibitor in a first cell culture medium to initiate the first expansion by culturing the first TIL population, where the AKT inhibitor is optionally selected from the group consisting of: pataxerti, GSK690693 , GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin A, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Magnolol, to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is initiated in a container comprising a first gas permeable surface area performing wherein the first expansion is initiated for a first period of about 1 to 7/8 days to obtain a second population of TILs, wherein the number of the second population of TILs is greater than that of the first population of TILs; (c) by including IL- 2. Cultivate the second TIL population in the second culture medium of OKT-3 and APC to carry out a rapid second expansion to produce a third TIL population, wherein the number of APCs added in the rapid second expansion is in step (b) at least twice the number of APCs added, wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein rapid second amplification is carried out in a container comprising a second gas-permeable surface area; (d) collecting the therapeutic TIL population obtained from step (d); (e) collecting the collected TIL population from step (e); transferring the TIL population to the infusion bag; and (f) optionally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time prior to the collecting step (e), such that the therapeutic TIL population comprises genetically modified Modified TILs, the genetically modified TILs comprise a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by dividing The obtained tumor sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from an individual's cancer; ) for an initial first expansion by culturing the first TIL population in a second cell culture medium to produce a second TIL population, wherein the initial first expansion is performed in a container comprising a first gas-permeable surface area, wherein the The initial first expansion is carried out for a first period of about 1 to 7/8 days to obtain the second TIL population, wherein the second TIL population is larger in number than the first TIL population; (c) by including IL-2 , OKT-3, APC and protein kinase B (AKT) inhibitors in the second culture medium of the second TIL population for rapid second expansion, wherein the AKT inhibitor is selected from the group consisting of: patase For, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin, Yellow acetin and honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the number of APCs added in the rapid second expansion is At least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain a therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population, wherein Rapid second expansion is carried out in a container comprising a second gas-permeable surface area; (d) collecting the therapeutic TIL population obtained from step (d); (e) collecting the collected TIL from step (e) the population is transferred to the infusion bag; and (f) optionally at any time prior to collecting step (e), genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population such that the therapeutic TIL population comprises the Genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by dividing The obtained tumor sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from an individual's cancer; ) and a protein kinase B (AKT) inhibitor in a first cell culture medium to initiate the first expansion by culturing the first TIL population, where the AKT inhibitor is optionally selected from the group consisting of: pataxerti, GSK690693 , GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin A, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Magnolol, to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is initiated in a container comprising a first gas permeable surface area performing wherein the first expansion is initiated for a first period of about 1 to 7/8 days to obtain a second population of TILs, wherein the number of the second population of TILs is greater than that of the first population of TILs; (c) by including IL- 2. Culture a second population of TILs in a second culture medium of OKT-3, APC and protein kinase B (AKT) inhibitors for rapid second expansion, where the AKT inhibitors are optionally selected from the group consisting of: Seti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin , luteinin and honokiol to produce a third TIL population, which is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the number of APCs added in the rapid second amplification is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain a therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population, wherein rapid second amplification is carried out in a container comprising a second gas-permeable surface area; (d) collecting the therapeutic TIL population obtained from step (d); (e) collecting the collected TIL population from step (e); transferring the TIL population to the infusion bag; and (f) optionally genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time prior to collecting step (e) such that the therapeutic TIL population comprises genetically modified TIL , the genetically modified TILs comprise a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by A tumor sample obtained from an individual is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual's or patient's cancer; (b) selecting CD39 ^LO from the first TIL population in (a) /CD69 ^LO and/or CD39/CD69 double-negative TIL to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population; (c) CD39 ^LO /CD69 ^LO and/or CD39/ The CD69 double negative TIL population is added to the closed system; (d) a first expansion is performed by culturing the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population enriched in IL-2 containing cell culture medium to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain a second population of TILs, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) by supplementing with additional IL-2, OKT-3 and antigen presenting cells (APCs) second expansion of the cell culture medium of the second TIL population to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second amplification is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) collecting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) is optionally carried out without opening the system; (g) converting the TIL population from step (f) The collected third TIL population is transferred to an infusion bag, wherein the transfer from steps (f) to (g) is optionally performed without opening the system; and (h) optionally during the collecting step (f) Enrichment of CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population and/or second TIL population and/or third TIL population such that the third TIL population comprises genetically modified TILs at any time prior to genetic modification , the genetically modified TILs comprise a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by processing a tumor sample obtained from an individual into a tumor digest to obtain and/or receive a first population of TILs derived from a tumor resected from an individual's or patient's cancer; Added to a closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from The group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, Nolide, isoliquiritigenin, chrysanthemum and honokiol to generate a second TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is based on The situation is carried out in a closed container providing a first gas-permeable surface area, wherein the first expansion is carried out for about 3-14 days to obtain a second TIL population, and wherein the transition from step (b) to step (c) is optionally at Performed without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen presenting cells (APCs) to generate a third TIL A population, wherein the second expansion is performed for about 7-14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally in a closed container providing a second gas-permeable surface area and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) collecting the third population of TILs obtained from step (d), wherein from step (d) ) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (e) to an infusion bag, wherein from step (e) The transfer to (f) is optionally performed without opening the system; and (g) optionally genetically modifying the first TIL population and/or the second TIL population and/or at any time prior to collecting step (f) or a third population of TILs such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by processing a tumor sample obtained from an individual into a tumor digest to obtain and/or receive a first population of TILs derived from a tumor resected from an individual's or patient's cancer; added to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is optionally at Provide a first gas permeable surface area in a closed container, wherein the first amplification is carried out for about 3-14 days to obtain the second TIL population, and wherein the transition from step (c) to step (d) is optionally at Performed without opening the system; (d) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors Second expansion, optionally wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifol New, oridonin, gossin, tenoride, isoliquiritigenin, chrysanthemum, and honokiol to generate a third TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/ CD69 double negative TIL population, wherein the second expansion is performed for about 7-14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally provided with a second gas permeable surface (e) collecting the third TIL population obtained from step (d), wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (e) to an infusion bag, wherein The transfer from steps (e) to (f) is optionally performed without opening the system; and (g) optionally at any time prior to collecting step (f) the genetically modified first TIL population and/or second The second population of TILs and/or the third population of TILs, such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by processing a tumor sample obtained from an individual into a tumor digest to obtain and/or receive a first population of TILs derived from a tumor resected from an individual's or patient's cancer; Added to a closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from The group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, Nolide, isoliquiritigenin, chrysanthemum and honokiol to generate a second TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is based on The situation is carried out in a closed container providing a first gas-permeable surface area, wherein the first expansion is carried out for about 3-14 days to obtain a second TIL population, and wherein the transition from step (b) to step (c) is optionally at (d) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors. Two expansions, optionally wherein the AKT inhibitor is selected from the group consisting of: paltaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine , Rubescensin A, Gossin, Tenolide, Isoliquiritigenin, Parcetrin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 Double negative TIL population, wherein the second expansion is performed for about 7-14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally provided in a second gas permeable surface area (e) collecting the third TIL population obtained from step (d), wherein The transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (e) to an infusion bag, wherein The transfer of steps (e) to (f) depends on whether performed with the system open; and (g) optionally genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time prior to collecting step (f) such that the third TIL population Included are genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by A tumor sample obtained from an individual is processed into a tumor digest to obtain a first TIL population derived from a tumor resected from the individual's cancer; b) selecting CD39 ^LO /CD69 ^LO and/or CD39 from the first TIL population in (a) /CD69 double negative TILs to obtain TIL populations enriched in (i) CD39 ^LO / CD69 ^LO and/or CD39/CD69 double negative; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative as appropriate The negative TIL population is added to the closed system; (d) a first expansion is performed by culturing the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population in cell culture medium containing IL-2 , to generate a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain a second population of TILs, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) by supplementing the first step with additional IL-2, OKT-3 and antigen presenting cells (APCs) The cell culture medium of two TIL populations is used to carry out second expansion, to produce the third TIL population, wherein second expansion carries out about 7-11 days to obtain the third TIL population, and wherein second expansion system is optionally in the provision of second carried out in a closed container with a gas-permeable surface area, and wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) collecting the third TIL obtained from step (e) A population, wherein the transition from step (e) to step (f) is optionally performed without opening the system; (g) transferring the collected third TIL population from step (f) to an infusion bag , wherein the transfer from steps (f) to (g) is optionally performed without opening the system; and (h) optionally genetically modified to enrich for CD39 ^LO /CD69 at any time prior to collecting step (f) ^LO and/or a CD39/CD69 double negative TIL population and/or a second TIL population and/or a third TIL population such that the third TIL population comprises genetically modified TILs comprising reduced CD39 and Genetic modification of CD69 expression.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by processing a tumor sample obtained from an individual into a plurality of tumor fragments or by processing a tumor sample obtained from the individual into a tumor digest to obtain a first population of TILs derived from tumors resected from the individual; (b) adding the tumor fragments or the tumor lysate, as appropriate, to the closed system; (c ) performing the first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: patase For, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin, Yellow acetin and honokiol to generate a second TIL population that is CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is optionally provided in the first gas permeable surface area wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally performed without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed About 7-11 days to obtain a third TIL population, wherein the second expansion is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally carried out without opening the system; (e) collecting the third TIL population obtained from step (e), wherein the transition from step (d) to step (e) is optionally without opening the system performing; (f) transferring the collected third population of TILs from step (f) to an infusion bag, wherein the transfer from steps (e) to (f) is optionally performed without opening the system; (g) cryopreserving the infusion bag from step (f) comprising the collected TIL population using a cryopreservation process; (h) administering to the individual a therapeutically effective dose of the third infusion bag from step (g). and (i) optionally genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time prior to administering step (h) such that the administered third TIL population Included are genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by processing a tumor sample obtained from an individual into a plurality of tumor fragments or by processing a tumor sample obtained from the individual into a tumor digest to obtain a first population of TILs derived from tumors resected from the individual; (b) adding the tumor fragments or the tumor lysate, as appropriate, to the closed system; (c ) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is optionally in an enclosure providing a first gas permeable surface area in a container, wherein the first amplification is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally performed without opening the system (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors, where appropriate The AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, Gossythin, tenolide, isoliquiritigenin, lutein, and honokiol to generate a third TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population, wherein The second expansion is performed for about 7-14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is carried out without opening the system as appropriate; (e) collecting the third TIL population obtained from step (e), wherein from step (d) to step The conversion of (e) is carried out without opening the system as appropriate; (f) transferring the collected third TIL population from step (f) to an infusion bag, wherein steps (e) to (f) ) is transferred optionally without opening the system; (g) cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h) administering to the individual a therapeutically effective dose of the third population of TILs from the infusion bag in step (g); and (i) optionally genetically modifying the first population of TILs and/or the second population of TILs at any time prior to administering step (h) and and/or a third population of TILs, such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by processing a tumor sample obtained from an individual into a plurality of tumor fragments or by processing a tumor sample obtained from the individual into a tumor digest to obtain a first population of TILs derived from tumors resected from the individual; (b) adding the tumor fragments or the tumor lysate, as appropriate, to the closed system; (c ) performing the first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: patase For, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin, Yellow acetin and honokiol to generate a second TIL population that is CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is optionally provided in the first gas permeable surface area wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally performed without opening the system; (d) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs), and protein kinase B (AKT) inhibitors, where AKT The inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, Gossin, tenolide, isoliquiritigenin, lutein and honokiol to generate the third TIL population, which is CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the third TIL population The second expansion is performed for about 7-14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein The transition from step (c) to step (d) is optionally carried out without opening the system; (e) collecting the third TIL population obtained from step (e), wherein from step (d) to step ( The conversion of e) is performed without opening the system as appropriate; (f) transferring the collected third TIL population from step (f) to an infusion bag, wherein steps (e) to (f) The transfer is carried out without opening the system as the case may be (g) cryopreserving the infusion bag from step (f) comprising the collected TIL population using a cryopreservation process; (h) administering to the individual a therapeutically effective dose of the first infusion bag from step (g) three TIL populations; and (i) optionally genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time prior to administering step (h) such that the third TIL population administered The population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by surgical resection, biopsy biopsy, core needle biopsy, mini biopsy or other Means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer in a patient or individual Obtaining and/or receiving a first TIL population, (b) selecting CD39 ^LO /CD69 from the first TIL population in (a) ^LO and/or CD39/CD69 double-negative TILs to obtain CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations; (c) enrich CD39 ^LO /CD69 ^LO and/or CD39/ The CD69 double negative TIL population is added to the closed system; (d) the first TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative is performed by culturing the TIL population enriched in IL-2 containing cell culture medium. Expansion to produce a second population of TILs, wherein the first expansion is optionally performed in a closed vessel providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs , and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) by adding additional IL-2, OKT-3 and antigen presenting cells (APC) Supplementing the cell culture medium of the second TIL population for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the second expansion is optionally provided The second air-permeable surface area is carried out in a closed container, and wherein the transition from step (d) to step (e) is optionally carried out without opening the system; (f) collecting the first gas obtained from step (e); Three TIL populations, wherein the transition from step (e) to step (f) is optionally carried out without opening the system; (g) transferring the collected third TIL population from step (f) to an infusion bag, wherein transfer from steps (e) to (f) is optionally performed without opening the system; and (h) genetically modified enriched CD39 ^LO is optionally collected at any time prior to step (f) /CD69 ^LO and/or CD39/CD69 double negative TIL population and/or second TIL population and/or third TIL population such that the third TIL population comprises genetically modified TILs comprising reduced Genetic modification of expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by surgical resection, biopsy biopsy, core needle biopsy, mini biopsy or other The means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer in the patient or individual obtains and/or receives a first TIL population, (b) optionally adding the tumor fragments or the tumor lysates into a closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of Constituent group: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifoxin, oridonin, cotton xanthin, tenol Lido, isoliquiritigenin, chrysanthemum and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative, where the first amplification is optional Carried out in a closed vessel providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain a second TIL population, and wherein the transition from step (b) to step (c) is optionally performed at different times In the case of an open system; (d) a second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen presenting cells (APCs) to generate a third TIL population , wherein the second amplification is performed for about 7-11 days to obtain a third TIL population, wherein the second amplification is optionally performed in a closed container providing a second gas-permeable surface area, and wherein from step (c) to step ( The transformation of d) is optionally carried out without opening the system; (e) collecting the third TIL population obtained from step (d), wherein the transformation from step (d) to step (e) is optionally carried out at (f) transferring the collected third TIL population from step (e) to an infusion bag, wherein the transfer from steps (e) to (f) is optionally performed without opening the system; and (g) optionally at any time prior to collecting step (e), genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population such that the third TIL population comprises Genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by surgical resection, biopsy biopsy, core needle biopsy, mini biopsy or other The means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer in the patient or individual obtains and/or receives a first TIL population, (b) optionally adding the tumor fragments or the tumor lysates to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is optionally provided Performed in a closed container of the first air-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally performed at different times (d) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors. Two expansions, optionally wherein the AKT inhibitor is selected from the group consisting of: paltaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine , Rubescensin A, Gossin, Tenolide, Isoliquiritigenin, Parcetrin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 Double negative TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally provided in a second gas permeable surface area (e) collecting the third TIL population obtained from step (d), wherein The transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (e) to an infusion bag, wherein The transfer of steps (e) to (f) is optionally performed without opening the system; and (g) optionally genetically modifying the first TIL population and/or the second population of TILs at any time prior to collecting step (e) The population of TILs and/or the third population of TILs such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by surgical resection, biopsy biopsy, core needle biopsy, mini biopsy or other Means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer in the patient or individual obtains and/or receives a first TIL population, (b) optionally adding tumor fragments or tumor digests to the closed system (c) performing the first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: Pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifoxine, Oridonin, Gossin, Tenolide, Isolide Liquiritigenin, luteinin, and honokiol to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplification is optionally provided in the first Performed in a closed container with a gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain a second TIL population, and wherein the transition from step (b) to step (c) is optionally without an open system (d) a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors, depending on In the case where the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonium Resin, Gossin, Tenolid, Isoliquiritigenin, Paretin, and Honokiol to generate a third TIL population that is CD39 ^LO /CD69 ^LO enriched and/or CD39/CD69 double negative TIL population , wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed vessel providing a second gas-permeable surface area , and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) collecting the third population of TILs obtained from step (d), wherein from step (d) The transition to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (e) to an infusion bag, wherein from step (e) to (f) The transfer is based on not opening the system as the case may be and (g) optionally genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time prior to collecting step (e) such that the third TIL population comprises genetically modified Modified TILs, the genetically modified TILs comprise a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) resecting a tumor from a cancer in an individual or patient, the tumor comprising a first TIL population , optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into multiple Tumor fragments or tumor digests; (c) enzymatically digesting multiple tumor fragments to obtain a first TIL population; (d) selecting CD39 ^LO /CD69 ^LO and/or CD39 from the first TIL population in (c) /CD69 double-negative TILs to obtain CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations; (e) Enrich CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TILs as appropriate The population is added to the closed system; (f) a first expansion is performed by culturing the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population in cell culture medium comprising IL-2 to produce A second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain a second population of TILs, and wherein from step (e) The transition to step (f) is performed optionally without opening the system; (g) by supplementing the second TIL population with additional IL-2, OKT-3 and antigen presenting cells (APCs) To produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, wherein the second expansion is optionally provided with a second gas-permeable surface area (h) collecting the third population of TILs obtained from step (g), wherein from The transition from step (g) to step (h) is performed optionally without opening the system; (i) transferring the collected third TIL population from step (h) to an infusion bag, wherein from step (h) ) to (i) transfer is optionally performed without opening the system; and (j) genetically modified to enrich for CD39 ^LO /CD69 ^LO and/or CD39/CD69 at any time prior to harvesting step (h) The double negative TIL population and/or the second TIL population and/or the third TIL population such that the third TIL population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) resecting a tumor from a cancer in an individual or patient, the tumor comprising a first TIL population , optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into multiple tumor fragments or tumor digests; (c) enzymatically digesting multiple tumor fragments to obtain a first TIL population; (d) optionally adding tumor fragments or tumor digests to the closed system; (e) by The first expansion is performed by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693 , GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin A, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Magnolol, to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is optionally in a closed container that provides a first gas permeable surface area wherein the first amplification is performed for about 3-11 days to obtain a second TIL population, and wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7- 11 days to obtain a third TIL population, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (e) to step (f) is optionally performed in a closed container (g) collecting the third TIL population obtained from step (f), wherein the transition from step (f) to step (g) is optionally performed without opening the system; (h) transferring the collected third TIL population from step (g) to an infusion bag, wherein the transfer from steps (g) to (h) is optionally performed without opening the system; and (i) optionally in At any time prior to collecting step (g), genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population such that the third TIL population comprises genetically modified TILs comprising Genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) resecting a tumor from a cancer in an individual or patient, the tumor comprising a first TIL population , optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into multiple tumor fragments or tumor digests; (c) enzymatically digesting multiple tumor fragments to obtain a first TIL population; (d) optionally adding tumor fragments or tumor digests to the closed system; (e) by A first expansion is performed by culturing the first TIL population in cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is optionally performed in a closed vessel providing a first gas permeable surface area , wherein the first amplification is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (e) to step (f) is optionally performed without opening the system; (f ) Second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors, optionally wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton yellow TILs enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TILs, wherein the second amplified The increase is carried out for about 7-11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein from step (e) the transition to step (f) is optionally carried out without opening the system; (g) collecting the third population of TILs obtained from step (f), wherein the transition from step (f) to step (g) The conversion is performed optionally without opening the system; (h) transferring the collected third TIL population from step (g) to an infusion bag, wherein the transfer from steps (g) to (h) is optional performed without opening the system; and (i) optionally genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time prior to collecting step (g) such that the third TIL The population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) resecting a tumor from a cancer in an individual or patient, the tumor comprising a first TIL population , optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into multiple tumor fragments or tumor digests; (c) enzymatically digesting multiple tumor fragments to obtain a first TIL population; (d) optionally adding tumor fragments or tumor digests to the closed system; (e) by The first expansion is performed by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693 , GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin A, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Magnolol, to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is optionally in a closed container that provides a first gas permeable surface area wherein the first amplification is performed for about 3-11 days to obtain a second TIL population, and wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs), and protein kinase B (AKT) inhibitors, optionally where the AKT inhibitor is selected from the group consisting of pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, resinthin , tenolid, isoliquiritigenin, lutein and honokiol to generate a third TIL population, which is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the second amplified for about 7-11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein steps from ( e) the transition to step (f) is optionally carried out without opening the system; (g) collecting the third TIL population obtained from step (f), wherein the transition from step (f) to step (g) Depending on the situation, between closed systems (h) transferring the collected third TIL population from step (g) to an infusion bag, wherein the transfer from steps (g) to (h) is optionally performed without opening the system; and (i) optionally at any time prior to collecting step (g), genetically modifying the first population of TILs and/or the second population of TILs and/or the third population of TILs such that the third population of TILs comprises genetically modified TILs, the Such genetically modified TILs include genetic modifications that reduce the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by surgical resection, biopsy biopsy, core needle biopsy, mini biopsy or other Means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer in an individual or patient Obtaining and/or receiving a first TIL population; (b) selecting CD39 ^LO /CD69 from the first TIL population in (a) ^LO and/or CD39/CD69 double-negative TILs to obtain CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations; (c) enrich CD39 ^LO /CD69 ^LO and/or CD39/CD69 double The negative TIL population is contacted with the first cell culture medium; (d) performing an initial expansion (or initiation of the first expansion) of a CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population in the first cell culture medium ) to obtain a second population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), wherein the first expansion is initiated expanding for a period of 1 to 8 days; (e) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT - 3 (anti-CD3 antibody) and APC; and wherein the rapid expansion is performed for a period of 14 days or less, optionally the rapid second amplification may be performed 1 day, 2 days after initiation of the rapid second amplification , 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days; (f) collecting the third TIL population; Temporal genetic modification enriches CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations and/or second TIL populations and/or third TIL populations such that the third TIL populations comprise genetically modified TILs, which Genetically modified TILs include genetic modifications that reduce the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by surgical resection, biopsy biopsy, core needle biopsy, mini biopsy or other The means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer in an individual or patient obtains and/or receives a first TIL population; (b) by adding IL-2, optionally OKT-3 (anti-CD3 antibody), optionally antigen-presenting cells (APCs), and protein kinase B (AKT) inhibitors in cell culture medium for initial expansion (or initiation of the first expansion) by culturing the first TIL population, depending on In the case where the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonium Resin, Gossythin, Tenolid, Isoliquiritigenin, Paretin, and Honokiol to generate a second TIL population that is CD39 ^LO /CD69 ^LO enriched and/or CD39/CD69 double negative TIL population , wherein the first amplification is optionally performed in a closed vessel providing a first gas-permeable surface area, wherein the initial first amplification is performed for about 1-8 days to obtain a second TIL population, and wherein from steps (a) to The conversion of step (b) is optionally performed without opening the system; (c) performing a rapid second amplification of the second TIL population in a second cell culture medium to obtain a third TIL population; wherein the first The two-cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and APCs; and wherein the rapid expansion is performed for a period of 14 days or less, optionally the rapid second expansion can be performed during the rapid second expansion 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation; (d) collecting a third TIL population; and (e) optionally at At any time prior to collecting step (d), genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population such that the third TIL population comprises genetically modified TILs comprising Genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by surgical resection, biopsy biopsy, core needle biopsy, mini biopsy or other Means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer in an individual or patient Obtaining and/or receiving a first TIL population; (b) performing an initial expansion of the first TIL population in a first cell culture medium (or initiate first expansion) to obtain a second population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen presenting cells (APCs) , wherein the initial first expansion is performed for a period of 1 to 8 days; (c) performing a rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT- 3 (anti-CD3 antibody), APC and protein kinase B (AKT) inhibitors, where appropriate, wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867 , CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, nossinin, tenoride, isoliquiritigenin, luteinin and honokiol to produce the third TIL population, which To enrich for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion can be performed within the rapid second expansion 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation; (d) collecting a third TIL population; and (e) optionally at At any time prior to collecting step (d), genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population such that the third TIL population comprises genetically modified TILs comprising Genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the following steps: (a) by surgical resection, biopsy biopsy, core needle biopsy, mini biopsy or other The means for obtaining a sample containing a mixture of tumor and TIL cells from a cancer in an individual or patient obtains and/or receives a first TIL population; (b) by adding IL-2, optionally OKT-3 (anti-CD3 antibody), optionally antigen-presenting cells (APCs), and protein kinase B (AKT) inhibitors in cell culture medium for initial expansion (or initiation of the first expansion) by culturing the first TIL population, depending on In the case where the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonium Resin, Gossythin, Tenolid, Isoliquiritigenin, Paretin, and Honokiol to generate a second TIL population that is CD39 ^LO /CD69 ^LO enriched and/or CD39/CD69 double negative TIL population , wherein the first amplification is optionally performed in a closed vessel providing a first gas-permeable surface area, wherein the initial first amplification is performed for about 1-8 days to obtain a second TIL population, and wherein from steps (a) to The conversion of step (b) is optionally performed without opening the system; (c) performing a rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), APC, and protein kinase B (AKT) inhibitors, where appropriate, wherein the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068 , AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenolid, isoliquiritigenin, luteinin and honokiol to generate the third TIL population , which is a population of TILs enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative, wherein the rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion can be performed within the rapid second 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of amplification; (d) collecting a third TIL population; and (e) depending on Situation At any time prior to collecting step (d), the first TIL population and/or the second TIL population and/or the third TIL population are genetically modified such that the third TIL population comprises genetically modified TILs that are genetically modified TILs contain genetic modifications that reduce the expression of CD39 and CD69 .

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the steps of: a) resecting a tumor from the cancer in the individual or patient, the tumor comprising the first TIL population , optionally by surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) fragmenting the tumor into multiple Tumor fragments or tumor digests; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs are selected from the first TIL population in tumor fragments or tumor digests to obtain enriched CD39 ^LO /CD69 ^LO and/ or a CD39/CD69 double-negative TIL population; (d) contacting a TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative with the first cell culture medium; (e) enriching in the first cell culture medium Collect initial expansion (or initiate first expansion) of CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally Optional OKT-3 (anti-CD3 antibody) and optionally antigen-presenting cells (APC), wherein the first expansion is initiated for a period of 1 to 8 days; (f) the second is performed in a second cell culture medium Rapid second expansion of the TIL population to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and APCs; and wherein the rapid expansion is performed for 14 days or less A period of time, optionally the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days; (g) collecting the third TIL population; and (h) genetically modifying CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations and/or at any time before collecting (f) as the case may be Or the second population of TILs and/or the third population of TILs, such that the third population of TILs comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the steps of: a) resecting a tumor from the cancer in the individual or patient, the tumor comprising the first TIL population , optionally by surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) fragmenting the tumor into multiple Tumor fragments or tumor digests; (c) obtained by combining IL-2, optionally OKT-3 (anti-CD3 antibody), optionally antigen presenting cells (APC) and protein kinase B (AKT) inhibitors Initial expansion (or initiation of the first expansion) is performed by culturing the first TIL population in cell culture medium, where the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363 , GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenoride, isoliquiritigenin, chrysanthemum and honokiol to produce A second TIL population, which is a TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative, wherein the first amplification is optionally performed in a closed container providing a first gas-permeable surface area, wherein the initial The first expansion is performed for about 1-8 days to obtain a second population of TILs, and wherein the transition from step (a) to step (b) is optionally performed without opening the system; (d) in the second cell performing a rapid second expansion of the second TIL population in culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and APC; and wherein the rapid expansion Performed for a period of 14 days or less, optionally the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days; (e) collecting the third TIL population; and (f) genetically modifying the first TIL population and/or the second TIL population and/or at any time prior to collecting (e) as the case may be A third population of TILs such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the steps of: a) resecting a tumor from the cancer in the individual or patient, the tumor comprising the first TIL population , optionally by surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) fragmenting the tumor into multiple tumor fragments or tumor digests; (c) performing an initial expansion (or initiating a first expansion) of a first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL- 2. Optionally select OKT-3 (anti-CD3 antibody) and optionally select antigen-presenting cells (APC), wherein the first expansion is initiated for a period of 1 to 8 days; (d) in the second cell culture medium A rapid second expansion is performed to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), APC, and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, resinthin , tenolid, isoliquiritigenin, luteinin, and honokiol to generate a third TIL population, which is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population, wherein rapid expansion was performed A period of about 14 days or less, optionally the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days; (e) collecting the third TIL population; and (f) genetically modifying the first TIL population and/or the second TIL population and/or at any time prior to collecting (e) as the case may be A third population of TILs such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the steps of: a) resecting a tumor from the cancer in the individual or patient, the tumor comprising the first TIL population , optionally by surgical resection, needle biopsy, core needle biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b) fragmenting the tumor into multiple Tumor fragments or tumor digests; (c) obtained by combining IL-2, optionally OKT-3 (anti-CD3 antibody), optionally antigen presenting cells (APC) and protein kinase B (AKT) inhibitors Initial expansion (or initiation of the first expansion) is performed by culturing the first TIL population in cell culture medium, where the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363 , GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenoride, isoliquiritigenin, chrysanthemum and honokiol to produce A second TIL population, which is a TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative, wherein the first amplification is optionally performed in a closed container providing a first gas-permeable surface area, wherein the initial The first expansion is performed for about 1-8 days to obtain a second population of TILs, and wherein the transition from step (a) to step (b) is optionally performed without opening the system; (d) in the second cell A rapid second expansion in culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), APC and protein kinase B (AKT) inhibitor, where AKT inhibits The agent is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, grass cotton flavin, tenolide, isoliquiritigenin, lutealin and honokiol to generate a third TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, in which rapidly expanding Amplification is performed for a period of about 14 days or less, optionally the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days after initiation of the rapid second amplification days, 8 days, 9 days or 10 days; (e) collecting the third TIL population; and (f) genetically modifying the first TIL population and/or the second TIL population at any time before collecting (e) as the case may be and /or a third population of TILs such that the third population of TILs comprises genetically modified Decorated TILs comprising genetic modifications that reduce the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by dividing The obtained tumor sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual's cancer; (b) selecting CD39 ^LO /CD69 ^LO from the first TIL population in step (a) and/or CD39/CD69 double-negative TILs to obtain a population of TILs enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negatives; (c) by including IL-2, optionally OKT-3 and An initial first expansion is performed by culturing a CD39/CD69 double negative TIL population enriched in cell culture medium optionally comprising antigen presenting cells (APCs) to generate a second TIL population, wherein the initial first expansion is performed for about A first period of 1 to 11 days to obtain a second TIL population, wherein the number of the second TIL population is greater than the first TIL population; (d) by making the second TIL population with IL-2, OKT-3 Contacting the cell culture medium of APCs to perform a rapid second expansion to produce a third TIL population, wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, wherein the third The TIL population is a therapeutic TIL population; (e) collecting the therapeutic TIL population obtained from step (c); and (f) optionally genetically modifying CD39 ^LO /CD69 ^LO at any time prior to collecting step (e) and /or a CD39/CD69 double negative TIL population and/or a second TIL population and/or a third TIL population such that the third TIL population comprises genetically modified TILs comprising reduced CD39 and CD69 Genetic modification of expression.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by dividing The obtained tumor sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from an individual's cancer; ) and a protein kinase B (AKT) inhibitor in a first cell culture medium to initiate the first expansion by culturing the first TIL population, where the AKT inhibitor is optionally selected from the group consisting of: pataxerti, GSK690693 , GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin A, Gossythin, Tenoride, Isoliquiritigenin, Huangpi and Magnolol, to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is initiated in a container comprising a first gas permeable surface area performing wherein the initial first expansion is performed for a first period of about 1 to 11 days to obtain a second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (c) by making the second population of TILs Contact with cell culture medium comprising IL-2, OKT-3, and APC to perform a rapid second expansion to produce a third TIL population, wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the a third TIL population, wherein the third TIL population is a therapeutic TIL population; (d) collecting the therapeutic TIL population obtained from step (c); and (e) optionally at any time prior to collecting step (d) The first TIL population and/or the second TIL population and/or the third TIL population are modified such that the third TIL population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by dividing The obtained tumor sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from an individual's cancer; (b) by including IL-2, optionally OKT-3 and optionally Situations comprising culturing the first population of TILs in cell culture medium of antigen presenting cells (APCs) for an initial first expansion to produce a second population of TILs, wherein the initial first expansion is performed for about 1 to 11 days A period of time to obtain a second TIL population, wherein the number of the second TIL population is greater than the first TIL population; (c) by making the second TIL population and comprising IL-2, OKT-3, APC and protein kinase B ( Rapid secondary expansion by exposure to cell culture medium of an AKT inhibitor, where appropriate, wherein the AKT inhibitor is selected from the group consisting of: paltaxet, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930 , MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenoride, isoliquiritigenin, luteinin and honokiol to produce the third TIL population, which is rich in Collect CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, where rapid second expansion is performed for a second period of about 1 to 11 days to obtain a third TIL population, where the third TIL population is a therapeutic TIL (d) collecting the therapeutic TIL population obtained from step (c); and (e) genetically modifying the first TIL population and/or the second TIL population and/or optionally at any time prior to collecting step (d) A third population of TILs such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by dividing The obtained tumor sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from an individual or patient's cancer; (APC) and a protein kinase B (AKT) inhibitor in the first cell culture medium to initiate the first expansion by culturing the first TIL population, where the AKT inhibitor is optionally selected from the group consisting of: pataxerti , GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin, Huangpi and honokiol to generate a second TIL population that is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplification is initiated in a region comprising the first gas permeable surface region carried out in a container wherein the initial first amplification is performed for a first period of about 1 to 11 days to obtain a second population of TILs, wherein the number of the second population of TILs is greater than that of the first population of TILs; (c) by making the second population of TILs The population is contacted with a second cell culture medium comprising IL-2, OKT-3, APC, and a protein kinase B (AKT) inhibitor for rapid second expansion, where the AKT inhibitor is optionally selected from the group consisting of: Pa Taserti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifoxin, Oridonin A, Gossin, Tenoride, Isolicorice Chlorin, lutein and honokiol to generate a third TIL population, which is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein rapid second expansion is performed for about 1 to 11 days a second period to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population; (d) collecting the therapeutic TIL population obtained from step (c); and (e) optionally prior to collecting step (d) Genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time such that the third TIL population comprises genetically modified TILs comprising reduced expression of CD39 and CD69 of genetic modification.

In some embodiments, the cell culture medium further comprises antigen presenting cells (APCs) during the initiation of the first expansion step, and wherein the number of APCs in the culture medium during the rapid second expansion step is greater than that of the initiation of the first expansion The number of APCs in the medium in the step.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) by surgical excision, biopsy biopsy, core needle biopsy, mini biopsy or other methods for Means of obtaining a sample containing a mixture of tumor and TIL cells from a cancer in a patient or individual obtains and/or receives a first TIL population, (b) selects CD39 ^LO /CD69 ^LO from the first TIL population in (a) and /or CD39/CD69 double-negative TIL to obtain enriched CD39 ^LO / CD69 ^LO and/or CD39/CD69 double-negative TIL population; (c) by including IL-2, OKT-3 and antigen-presenting cells (APC ) in the first cell culture medium enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population to initiate the first expansion to generate the second TIL population, wherein the initial first expansion is performed in a vessel comprising a first gas-permeable surface area, wherein the first expansion is initiated for a first period of about 1 to 11 days to obtain a second population of TILs, wherein the number of the second population of TILs is greater than that of the first population of TILs; ( d) optionally restimulating the second TIL population with OKT-3; (e) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a protein that reduces the expression of CD39 and CD69 genetic modification; (f) rapid second expansion by culturing the modified second TIL population in a second medium comprising IL-2, OKT-3, and APCs to produce a third TIL population, wherein the rapid second TIL population The second expansion is performed for a second period of about 14 days or less to obtain a therapeutic TIL population, wherein a third TIL population is a therapeutic TIL population comprising genetic modifications that reduce the expression of CD39 and CD69; and (g) collecting a third TIL population TIL groups.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) by surgical excision, biopsy biopsy, core needle biopsy, mini biopsy or other methods for A means of obtaining a sample containing a mixture of tumor and TIL cells from a cancer in a patient or individual to obtain and/or receive a first population of TILs, (b) by including IL-2, OKT-3, antigen presenting cells (APC ) and a protein kinase B (AKT) inhibitor in a first cell culture medium to initiate the first expansion by culturing the first TIL population, where the AKT inhibitor is optionally selected from the group consisting of: pataxerti, GSK690693 , GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin A, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Magnolol, to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is initiated in a container comprising a first gas permeable surface area proceeding wherein the first expansion is initiated for a first period of about 1 to 11 days to obtain a second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) optionally restimulated with OKT-3 a second TIL population; (d) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces the expression of CD39 and CD69; (e) by including Rapid second expansion of the modified second TIL population by culturing in the second medium of IL-2, OKT-3, and APC to generate a third TIL population, wherein the rapid second expansion is performed for about 14 days or less The second period of time is to obtain a therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population comprising genetic modifications that reduce the expression of CD39 and CD69; and (f) collecting the third TIL population.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) by surgical excision, biopsy biopsy, core needle biopsy, mini biopsy or other methods for A means of obtaining a sample containing a mixture of tumor and TIL cells from a cancer in a patient or individual to obtain and/or receive a first TIL population, (b) by including IL-2, OKT-3 and antigen presenting cells (APC ) to perform an initial first expansion by culturing the first TIL population in a second cell culture medium to produce a second TIL population, wherein the initial first expansion is carried out in a vessel comprising a first gas-permeable surface area, wherein the initial first expansion is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is larger in number than the first TIL population; (c) optionally with OKT-3 re-stimulating the second TIL population; (d) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces the expression of CD39 and CD69; (e) by A rapid second expansion is performed by culturing the modified second TIL population in a second medium comprising IL-2, OKT-3, APC, and a protein kinase B (AKT) inhibitor, where the AKT inhibitor is optionally selected from The group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, Nolide, isoliquiritigenin, chrysanthemum and honokiol to generate a third TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population where rapid second amplification was performed A second period of about 14 days or less to obtain a therapeutic TIL population, wherein a third TIL population is a therapeutic TIL population comprising a genetic modification that reduces the expression of CD39 and CD69; and (f) collecting the third TIL population.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) by surgical excision, biopsy biopsy, core needle biopsy, mini biopsy or other methods for A means of obtaining a sample containing a mixture of tumor and TIL cells from a cancer in a patient or individual to obtain and/or receive a first population of TILs, (b) by including IL-2, OKT-3, antigen presenting cells (APC ) and a protein kinase B (AKT) inhibitor in a first cell culture medium to initiate the first expansion by culturing the first TIL population, where the AKT inhibitor is optionally selected from the group consisting of: pataxerti, GSK690693 , GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin A, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Magnolol, to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is initiated in a container comprising a first gas permeable surface area proceeding wherein the first expansion is initiated for a first period of about 1 to 11 days to obtain a second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) optionally restimulated with OKT-3 a second TIL population; (d) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces the expression of CD39 and CD69; (e) by including Rapid second expansion by culturing the modified second TIL population in a second medium of IL-2, OKT-3, APC, and protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of The group: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, tenoli De, isoliquiritigenin, chrysanthemum and honokiol to generate a third TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations in which a rapid second amplification was performed for approximately 14 A second period of days or less to obtain a therapeutic TIL population, wherein a third TIL population is a therapeutic TIL population comprising a genetic modification that reduces the expression of CD39 and CD69; and (f) collecting the third TIL population.

In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, human papillomavirus Caused cancer, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer and renal cell carcinoma.

The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by adding IL-2, optionally OKT-3, and optionally A TIL population enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO is cultured in cell culture medium of antigen presenting cells (APCs) to perform the initial first expansion to generate a second TIL population, wherein the initial TIL population An expansion is performed for a first period of about 1 to 11 days to obtain a second population of TILs, wherein the second population of TILs is greater in number than the first population of TILs; (b) by combining the second population of TILs with 2. Contacting the cell culture medium of OKT-3 and APC to perform a rapid second expansion to produce a third TIL population, wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population , wherein the third TIL population is a therapeutic TIL population; and (c) collecting the third TIL population obtained from step (b); (d) genetically modifying CD39/CD69 at any time prior to collecting step (c) Double negative and/or CD39 ^LO /CD69 ^LO TIL population, second TIL population and/or third TIL population such that the collected third TIL population comprises genetically modified TILs comprising reduced Genetic modification of expression of CD39 and CD69.

In some embodiments, in step (a), the cell culture medium further comprises antigen-presenting cells (APCs), and wherein the number of APCs in the medium in step (c) is greater than the number of APCs in the medium in step (b) number.

A method for expanding T cells comprising: (a) initiating a first TIL population obtained from a donor by culturing the first TIL population to effect growth and initiate activation of the first T cell population an expansion wherein the first TIL population is enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL populations; (b) after activation of the first TIL population initiated in step (a) begins to decay , performing a rapid second expansion of the first TIL population by culturing the first TIL population to achieve growth and enhance activation of the first T cell population to obtain a second T cell population; (c) collecting the second T cell population population; and (d) genetically modifying the first population of TILs and/or the second population of TILs such that the collected second population of TILs comprises genetically modified TILs comprising reduced expression of CD39 and CD69 genetic modification.

A method for expanding T cells comprising: (a) priming a first T cell population from a tumor sample by culturing the first T cell population to effect growth and initiate activation of the first T cell population Initial first expansion, the tumor sample is obtained from one or more mini-biopsy, core-needle biopsy or needle biopsy of the tumor in the donor, where the first T cell population is enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO T cell population; (b) growth and enhancement of the second T cell population by culturing the first T cell population after activation of the first T cell population initiated in step (a) has begun to decay activation of a T cell population to perform rapid second expansion of the first T cell population to obtain a second T cell population; and (c) collecting the second T cell population; and (d) genetically modifying the first T cell population The cell population and/or the second TIL population such that the collected second T cell population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

In some embodiments, the second population of TILs from the first expansion or the third population of TILs from the second expansion, or both, are modified.

In some embodiments, the second population of TILs from the initial first expansion or the third population of TILs from the rapid second expansion, or both, are modified.

In some embodiments, the modification is performed on the second population of TILs from the first amplification and prior to the second amplification.

In some embodiments, modifications are made to the second population of TILs from the initial first expansion and prior to the rapid second expansion, or both.

In some embodiments, the third TIL population from the second expansion is modified.

In some embodiments, the third TIL population from the rapid second expansion is modified.

In some embodiments, modification is performed after collection.

In some embodiments, the first amplification is performed for a period of about 11 days.

In some embodiments, the first amplification is initiated for a period of about 11 days.

In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1000 IU/mL and 6000 IU/mL in the first expansion.

In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1000 IU/mL and 6000 IU/mL in the initial first expansion.

In some embodiments, in the second amplification step, IL-2 is present at an initial concentration of between 1000 IU/mL and 6000 IU/mL and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL.

In some embodiments, in the rapid second amplification step, IL-2 is present at an initial concentration of between 1000 IU/mL and 6000 IU/mL and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL.

In some embodiments, the first amplification is performed using a gas permeable container.

In some embodiments, the initial first amplification is performed using a gas permeable container.

In some embodiments, the second amplification is performed using a gas permeable container.

In some embodiments, the rapid second amplification is performed using a gas permeable container.

In some embodiments, the first expanded cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In some embodiments, the cell culture medium initiating the first expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In some embodiments, the second expanded cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In some embodiments, the rapid second expanded cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In some embodiments, the methods further comprise the step of treating the patient with a non-myeloablative lymphodepleting regimen prior to administering the third TIL population to the patient.

In some embodiments, the nonmyeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days, followed by 25 mg/m2/day Fludarabine was dosed for three days.

In some embodiments, the nonmyeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and cyclophosphamide at 25 mg/m2/day for two consecutive days. Fludarabine was dosed, followed by fludarabine at a dose of 25 mg/m2/day for three days.

In some embodiments, the nonmyeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and cyclophosphamide at 25 mg/m2/day for two consecutive days. Fludarabine was dosed, followed by fludarabine at a dose of 25 mg/m2/day for one day.

In some embodiments, cyclophosphamide is administered with mesna.

In some embodiments, the methods further comprise the step of starting treating the patient with the IL-2 regimen the day after administering the TIL to the patient.

In some embodiments, the method further comprises the step of initiating treatment of the patient with the IL-2 regimen on the same day as the TIL is administered to the patient.

In some embodiments, the IL-2 regimen is a low-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg aldesleukin or a biosimilar or variant thereof administered intravenously as a 15-minute bolus every eight hours Administer as an infusion until tolerated.

In some embodiments, a therapeutically effective TIL population is administered and the TIL population comprises about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ TILs.

In some embodiments, the initial first amplification and rapid second amplification are performed for a period of 21 days or less.

In some embodiments, the initial first amplification and rapid second amplification are performed for a period of 16 or 17 days or less.

In some embodiments, the first amplification is initiated for a period of 7 or 8 days or less.

In some embodiments, the rapid second amplification is performed for a period of 11 days or less.

In some embodiments, the first amplification and the second amplification are each performed separately over a period of 11 days.

In some embodiments, steps (a) to (f) are performed within about 26 days.

In some embodiments, the genetically modified TIL further comprises an additional genetic modification that reduces the expression of one or more of the following immune checkpoint genes selected from the group comprising: CTLA-4, LAG-3, HAVCR2 (TIM -3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3 and TOX.

In some embodiments, the one or more immune checkpoint genes are selected from the group comprising: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA.

In some embodiments, the modified TIL further comprises an additional genetic modification that results in enhanced expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population, the one or more immune checkpoint genes selected from the group consisting of Groups: CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1/2 intracellular domain (ICD) and / or NOTCH ligand mDLL1.

In some embodiments, the genetic modification step is performed using a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks at the one or more immune checkpoint genes.

In some embodiments, genetic modification is performed using one or more methods selected from the group consisting of CRISPR methods, TALE methods, zinc finger methods, and combinations thereof.

In some embodiments, the methods comprise CRISPR methods.

In some embodiments, the CRISPR method is a CRISPR/Cas9 method.

In some embodiments, genetic modification comprises TALE methods.

In some embodiments, the genetic modification comprises a zinc finger approach.

In some embodiments, processing a tumor sample obtained from an individual into a tumor digest comprises incubating the tumor sample in an enzymatic medium.

In some embodiments, processing the tumor sample obtained from the individual into a tumor digest further comprises mechanically disrupting the tumor sample to dissociate the tumor sample.

In some embodiments, processing the tumor sample obtained from the individual into a tumor digest further comprises purifying the dissociated tumor sample using density gradient separation.

In some embodiments, the enzyme medium comprises DNase.

In some embodiments, the enzyme medium comprises 30 units/ml of DNase.

In some embodiments, the enzyme medium comprises collagenase.

In some embodiments, the enzyme medium comprises 1.0 mg/mL of collagenase.

In some embodiments, the collected population of therapeutic TILs comprises sufficient TILs to administer a therapeutically effective dose to an individual.

In some embodiments, the therapeutically effective dose comprises about 1×10 ⁹ to about 9×10 ¹⁰ TILs.

In some embodiments, the APCs comprise peripheral blood mononuclear cells (PBMCs).

In some embodiments, the therapeutic TIL population collected in step (e) exhibits an increased subset of CD8+ cells compared to the first and/or second TIL population.

In some embodiments, PBMCs are supplemented at a ratio of about 1:25 TIL:PBMCs.

In some embodiments, the first amplification of the step and the second amplification of the step are each performed separately within a period of 11-12 days.

In some embodiments, steps (a) to (e), (f) or (g) are performed within about 10 days to about 24 days.

In some embodiments, steps (a) to (e), (f) or (g) are performed within about 15 days to about 24 days.

In some embodiments, steps (a) to (e), (f) or (g) are performed within about 20 days to about 24 days.

In some embodiments, steps (a) to (e), (f) or (g) are performed within about 20 days to about 22 days.

In some embodiments, the second TIL population is at least 50-fold greater in number than the first TIL population.

The invention also provides a population of TILs according to any of the methods described herein.

The invention also provides compositions comprising a population of TILs according to any of the methods described herein.

Brief Description of the Sequence Listing

SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

SEQ ID NO: 2 is the amino acid sequence of the light chain of murozumab.

SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

SEQ ID NO:5 is a form of IL-2.

SEQ ID NO: 6 is the amino acid sequence of nemvaleukin alfa.

SEQ ID NO: 7 is a form of IL-2.

SEQ ID NO: 8 is a mucin domain polypeptide.

SEQ ID NO:9 is the amino acid sequence of recombinant human IL-4 protein.

SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

SEQ ID NO: 13 is the IL-2 sequence.

SEQ ID NO: 14 is the IL-2 mutein sequence.

SEQ ID NO: 15 is the IL-2 mutein sequence.

SEQ ID NO: 16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

SEQ ID NO: 17 is the HCDR2 of IgG.IL2R67A.H1.

SEQ ID NO: 18 is the HCDR3 of IgG.IL2R67A.H1.

SEQ ID NO: 19 is the HCDR1_IL-2 kabat of IgG.IL2R67A.H1.

SEQ ID NO: 20 is the HCDR2 kabat of IgG.IL2R67A.H1.

SEQ ID NO: 21 is the HCDR3 kabat of IgG.IL2R67A.H1.

SEQ ID NO: 22 is HCDR1_IL-2 clothia of IgG.IL2R67A.H1.

SEQ ID NO: 23 is the HCDR2 clothia of IgG.IL2R67A.H1.

SEQ ID NO: 24 is the HCDR3 clothia of IgG.IL2R67A.H1.

SEQ ID NO: 25 is the HCDR1_IL-2 IMGT of IgG.IL2R67A.H1.

SEQ ID NO: 26 is the HCDR2 IMGT of IgG.IL2R67A.H1.

SEQ ID NO: 27 is the HCDR3 IMGT of IgG.IL2R67A.H1.

SEQ ID NO: 28 is the _VH chain of IgG.IL2R67A.H1.

SEQ ID NO: 29 is the heavy chain of IgG.IL2R67A.H1.

SEQ ID NO: 30 is the LCDR1 kabat of IgG.IL2R67A.H1.

SEQ ID NO: 31 is the LCDR2 kabat of IgG.IL2R67A.H1.

SEQ ID NO: 32 is the LCDR3 kabat of IgG.IL2R67A.H1.

SEQ ID NO: 33 is the LCDR1 chothia of IgG.IL2R67A.H1.

SEQ ID NO: 34 is the LCDR2 chothia of IgG.IL2R67A.H1.

SEQ ID NO: 35 is the LCDR3 chothia of IgG.IL2R67A.H1.

SEQ ID NO: 36 is the _VL chain.

SEQ ID NO: 37 is the light chain.

SEQ ID NO: 38 is the light chain.

SEQ ID NO: 39 is the light chain.

SEQ ID NO:40 is the amino acid sequence of human 4-1BB.

SEQ ID NO:41 is the amino acid sequence of murine 4-1BB.

SEQ ID NO: 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO: 43 is the light chain of the 4-1BB agonist monoclonal antibody utumumab (PF-05082566).

SEQ ID NO:44 is the heavy chain variable region (V _H ) of the 4-1BB agonist monoclonal antibody utumumab (PF-05082566).

SEQ ID NO:45 is the light chain variable region (V _L ) of the 4-1BB agonist monoclonal antibody utumumab (PF-05082566).

SEQ ID NO: 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utumumab (PF-05082566).

SEQ ID NO: 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utumumab (PF-05082566).

SEQ ID NO: 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utumumab (PF-05082566).

SEQ ID NO:49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utumumab (PF-05082566).

SEQ ID NO: 50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utumumab (PF-05082566).

SEQ ID NO:51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utumumab (PF-05082566).

SEQ ID NO:52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (urelumab) (BMS-663513).

SEQ ID NO:53 is the light chain of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513).

SEQ ID NO:54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513).

SEQ ID NO:55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513).

SEQ ID NO:56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513).

SEQ ID NO:57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513).

SEQ ID NO:58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513).

SEQ ID NO:59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513).

SEQ ID NO: 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513).

SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513).

SEQ ID NO: 62 is the Fc domain of a TNFRSF agonist fusion protein.

SEQ ID NO:63 is the linker of TNFRSF agonist fusion protein.

SEQ ID NO:64 is the linker of TNFRSF agonist fusion protein.

SEQ ID NO:65 is the linker of TNFRSF agonist fusion protein.

SEQ ID NO: 66 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 67 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 68 is the linker of TNFRSF agonist fusion protein.

SEQ ID NO: 69 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 70 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 71 is the linker of TNFRSF agonist fusion protein.

SEQ ID NO: 72 is the linker of TNFRSF agonist fusion protein.

SEQ ID NO: 73 is the Fc domain of a TNFRSF agonist fusion protein.

SEQ ID NO: 74 is the linker of TNFRSF agonist fusion protein.

SEQ ID NO: 75 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 76 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO:77 is the amino acid sequence of 4-1BB ligand (4-1BBL).

SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

SEQ ID NO:79 is the heavy chain variable region ( _VH ) of version 1 of 4-1BB agonist antibody 4B4-1-1.

SEQ ID NO: 80 is the light chain variable region (V _L ) of version 1 of 4-1BB agonist antibody 4B4-1-1.

SEQ ID NO: 81 is the heavy chain variable region (V _H ) of 4-1BB agonist antibody 4B4-1-1 version 2.

SEQ ID NO: 82 is the light chain variable region (V _L ) of 4-1BB agonist antibody 4B4-1-1 version 2.

SEQ ID NO: 83 is the heavy chain variable region (V _H ) of 4-1BB agonist antibody H39E3-2.

SEQ ID NO: 84 is the light chain variable region (V _L ) of 4-1BB agonist antibody H39E3-2.

SEQ ID NO:85 is the amino acid sequence of human OX40.

SEQ ID NO:86 is the amino acid sequence of murine OX40.

SEQ ID NO: 87 is the heavy chain of the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO: 88 is the light chain of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562).

SEQ ID NO:89 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562).

SEQ ID NO:90 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562).

SEQ ID NO: 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562).

SEQ ID NO:92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562).

SEQ ID NO:93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562).

SEQ ID NO: 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562).

SEQ ID NO:95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562).

SEQ ID NO:96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562).

SEQ ID NO:97 is the heavy chain of OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 98 is the light chain of OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 99 is the heavy chain variable region (V _H ) of OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 100 is the light chain variable region (V _L ) of OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 101 is the heavy chain CDR1 of OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 102 is the heavy chain CDR2 of OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 103 is the heavy chain CDR3 of OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 104 is the light chain CDR1 of OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 105 is the light chain CDR2 of OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 106 is the light chain CDR3 of OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 107 is the heavy chain of OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 108 is the light chain of OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 109 is the heavy chain variable region (V _H ) of OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 110 is the light chain variable region (V _L ) of OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 111 is the heavy chain CDR1 of OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 112 is the heavy chain CDR2 of OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 113 is the heavy chain CDR3 of OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 114 is the light chain CDR1 of OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 115 is the light chain CDR2 of OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 116 is the light chain CDR3 of OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 117 is the heavy chain variable region (V _H ) of OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 118 is the light chain variable region (V _L ) of OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 122 is the light chain CDR1 of OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 124 is the light chain CDR3 of OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 125 is the heavy chain variable region (V _H ) of OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 126 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 127 is the heavy chain CDR1 of OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 130 is the light chain CDR1 of OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 132 is the light chain CDR3 of OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 133 is the amino acid sequence of OX40 ligand (OX40L).

SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

SEQ ID NO: 136 is the heavy chain variable region (V _H ) of OX40 agonist monoclonal antibody 008.

SEQ ID NO: 137 is the light chain variable region (V _L ) of OX40 agonist monoclonal antibody 008.

SEQ ID NO: 138 is the heavy chain variable region (V _H ) of OX40 agonist monoclonal antibody 011.

SEQ ID NO: 139 is the light chain variable region (V _L ) of OX40 agonist monoclonal antibody 011.

SEQ ID NO: 140 is the heavy chain variable region (V _H ) of OX40 agonist monoclonal antibody 021.

SEQ ID NO: 141 is the light chain variable region (V _L ) of OX40 agonist monoclonal antibody 021.

SEQ ID NO: 142 is the heavy chain variable region (V _H ) of OX40 agonist monoclonal antibody 023.

SEQ ID NO: 143 is the light chain variable region (V _L ) of OX40 agonist monoclonal antibody 023.

SEQ ID NO: 144 is the heavy chain variable region (V _H ) of an OX40 agonist monoclonal antibody.

SEQ ID NO: 145 is the light chain variable region (V _L ) of an OX40 agonist monoclonal antibody.

SEQ ID NO: 146 is the heavy chain variable region (V _H ) of an OX40 agonist monoclonal antibody.

SEQ ID NO: 147 is the light chain variable region (V _L ) of an OX40 agonist monoclonal antibody.

SEQ ID NO: 148 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 149 is the heavy chain variable region ((V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 150 is the light chain variable region (V _L ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 151 is the light chain variable region (V _L ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 152 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 153 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 154 is the light chain variable region (V _L ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 155 is the light chain variable region (V _L ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 156 is the heavy chain variable region (V _H ) of an OX40 agonist monoclonal antibody.

SEQ ID NO: 157 is the light chain variable region (V _L ) of an OX40 agonist monoclonal antibody.

SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO: 160 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-1 inhibitor nivolumab.

SEQ ID NO: 161 is the amino acid sequence of the light chain variable region (V _L ) of the PD-1 inhibitor nivolumab.

SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO: 170 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-1 inhibitor pembrolizumab.

SEQ ID NO: 171 is the amino acid sequence of the light chain variable region (V _L ) of the PD-1 inhibitor pembrolizumab.

SEQ ID NO: 172 is the amino acid sequence of the heavy chain CDR1 of the PD-1 inhibitor pembrolizumab.

SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO: 180 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-L1 inhibitor durvalumab.

SEQ ID NO: 181 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor durvalumab.

SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO:183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO:190 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-L1 inhibitor avelumab.

SEQ ID NO: 191 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor avelumab.

SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

SEQ ID NO: 198 is the heavy chain amino acid sequence of PD-L1 inhibitor atezolizumab (atezolizumab).

SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:200 is the amino acid sequence of the heavy chain variable region (V _H ) of the PD-L1 inhibitor atezolizumab.

SEQ ID NO: 201 is the amino acid sequence of the light chain variable region (V _L ) of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:202 is the amino acid sequence of heavy chain CDR1 of PD-L1 inhibitor atezolizumab (atezolizumab).

SEQ ID NO:203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO:207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO: 209 is the light chain amino acid sequence of CTLA-4 inhibitor ipilimumab.

SEQ ID NO: 210 is the amino acid sequence of the heavy chain variable region (V _H ) of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO: 211 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO: 212 is the amino acid sequence of the heavy chain CDR1 of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

SEQ ID NO: 218 is the heavy chain amino acid sequence of CTLA-4 inhibitor tremelimumab.

SEQ ID NO: 219 is the light chain amino acid sequence of CTLA-4 inhibitor tremelimumab.

SEQ ID NO: 220 is the amino acid sequence of the heavy chain variable region (V _H ) of CTLA-4 inhibitor tremelimumab.

SEQ ID NO: 221 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO: 222 is the amino acid sequence of heavy chain CDR1 of CTLA-4 inhibitor tremelimumab.

SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

SEQ ID NO: 229 is the light chain amino acid sequence of CTLA-4 inhibitor Zefelizumab.

SEQ ID NO: 230 is the amino acid sequence of the heavy chain variable region (V _H ) of the CTLA-4 inhibitor Zefelizumab.

SEQ ID NO:231 is the amino acid sequence of the light chain variable region (V _L ) of the CTLA-4 inhibitor Zefelizumab.

SEQ ID NO: 232 is the amino acid sequence of the heavy chain CDR1 of the CTLA-4 inhibitor Zefelizumab.

SEQ ID NO: 233 is the amino acid sequence of the heavy chain CDR2 of the CTLA-4 inhibitor Zefelizumab.

SEQ ID NO:234 is the amino acid sequence of the heavy chain CDR3 of the CTLA-4 inhibitor Zefelizumab.

SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor Zefelizumab.

SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor Zefelizumab.

SEQ ID NO:237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor Zefelizumab. I. Introduction

Provided herein are TILs that are (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout (eg, genetically modified to silence or reduce CD39/CD69 expression), or (iii) a combination of (i) and (ii). In some embodiments, TILs of the invention are produced by genetically manipulating a population of TILs selected for: (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 dual genes Knockout (eg, genetically modified to silence or reduce CD39/CD69 expression), or (iii) a combination of (i) and (ii). Also provided herein are expansion methods for producing such genetically modified TILs and therapeutic methods using such TILs. Also provided herein are expansion methods for producing such genetically modified TILs and therapeutic methods using such TILs.

definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are hereby incorporated by reference in their entirety.

As used herein, the terms "co-administration", "co-administering", "administered in combination with", "combined with ... (administering in combination with)", "simultaneously" and "concurrently" encompass the administration of two or more active pharmaceutical ingredients (in some embodiments of the invention, such as a plurality of TILs) to an individual such that the two active pharmaceutical ingredients The constituents and/or their metabolites are also present in the individual. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.

The term "in vivo" refers to an event that occurs within the body of a subject.

The term "in vitro" refers to events that occur outside the body of a subject. In vitro assays encompass cell-based assays employing live or dead cells, and may also encompass cell-free assays that do not employ intact cells.

The term "ex vivo" refers to an event involving a treatment or procedure performed on cells, tissues and/or organs that have been removed from the body of an individual. Suitably, cells, tissues and/or organs may be returned to the individual using surgical or therapeutic methods.

The term "rapid expansion" means that the number of antigen-specific TILs increases by at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold or 9-fold) within a period of one week, more preferably within a period of one week Increase at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) within a period of time, or optimally at least about 100-fold within a one-week period. Various rapid amplification protocols are described herein.

"Tumor infiltrating lymphocytes" or "TILs" herein means a population of cells initially obtained as white blood cells that have left an individual's bloodstream and migrated into tumors. TILs include, but are not limited to, CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and secondary TILs. "Primary TILs" are cells obtained (sometimes referred to as "freshly collected") from a patient tissue sample as outlined herein, and "secondary TILs" are any population of TIL cells that has been expanded or proliferated as discussed herein, These include, but are not limited to, host TILs and expanded TILs ("REP TILs") or "post-REP TILs"). A population of TIL cells can include genetically modified TILs.

TILs can generally be defined biochemically (using cell surface markers) or functionally (by their ability to infiltrate tumors and effect therapy). TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors after reintroduction into a patient. TIL can be further characterized by potency, for example, if, for example, interferon (IFN) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL, then TIL is considered potent. If, for example, interferon (IFNγ) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL, greater than about 300 pg/mL, greater than about 400 pg/mL, Greater than about 500 pg/mL, greater than about 600 pg/mL, greater than about 700 pg/mL, greater than about 800 pg/mL, greater than about 900 pg/mL, greater than about 1000 pg/mL, TILs can be considered potent of.

"CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL" or "CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population" or a grammatical variation of any of the foregoing means the same as used in A TIL or TIL population that exhibits undetectable, lower or reduced mean cell surface protein CD39 and CD69 levels compared to any TIL/TIL population for which a reference TIL or TIL population is obtained.

"TIL enriched in CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO " or "TIL population enriched in CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO " or a grammatical variation of any of the foregoing By format is meant a TIL-enriched TIL or population of TILs with undetectable, low or reduced levels of mean cell surface proteins CD39 and CD69 compared to any TIL/TIL population used to obtain the reference TIL or population of TILs. Any enrichment means can be used to obtain enriched CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs, including sorting or selection for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs.

A "cell population" (including TILs) herein means a number of cells with common characteristics. Generally, population numbers range from 1×10 ⁶ to 1×10 ¹⁰ , with different TIL populations comprising different numbers. For example, initial growth of primary TILs in the presence of IL-2 produces a bulk TIL population of approximately 1 x ¹⁰⁸ cells. REP expansion is generally performed to provide a 1.5 x ¹⁰⁹ to 1.5 x ¹⁰¹⁰ cell population for infusion.

"Cryopreserved TIL" herein means processing and storing TIL, whether primary, bulk or expanded (REP TIL), within the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that can be used as a source of primary TILs.

Herein "thawed cryopreserved TILs" means a population of TILs that were previously cryopreserved and subsequently processed to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures at which TILs can be administered to a patient.

TILs can generally be defined biochemically (using cell surface markers) or functionally (by their ability to infiltrate tumors and effect therapy). TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors after reintroduction into a patient.

The term "cryopreservation media/cryopreservation medium" refers to any medium that can be used for cryopreservation of cells. Such media may include media comprising 7% to 10% DMSO. Exemplary media include CryoStor CS10, HypoThermosol, and combinations thereof. The term "CS10" refers to cryopreservation medium obtained from Stemcell Technologies or Biolife Solutions. The CS10 medium can be referred to by the trade name "CryoStor®CS10". CS10 Medium is a serum-free, animal component-free medium containing DMSO.

The term "central memory T cells" refers to a subset of T cells that in humans are CD45R0+ and constitutively express CCR7 (CCR7 ^hi ) and CD62L (CD62 ^hi ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2 and BMI1. Central memory T cells mainly secrete IL-2 and CD40L as effector molecules after TCR priming. Central memory T cells are found predominantly in the CD4 compartment of blood and are proportionally enriched in lymph nodes and tonsils in humans.

The term "effector memory T cells" refers to a subset of human or mammalian T cells, such as central memory T cells, that are CD45R0+ but have lost constitutive expression of CCR7 (CCR7 ^lo ) and are heterogeneous for CD62L expression or Low (CD62L ^lo ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines after antigen stimulation, including interferon-γ, IL4- and IL-5. Effector memory T cells are predominantly found in the CD8 compartment of the blood and are proportionally enriched in the lung, liver and gut in humans. CD8+ effector memory T cells carry high amounts of perforin.

The term "closed system" means a system that is closed from the external environment. Any closed system suitable for cell culture methods can be used in the methods of the invention. Closed systems include, for example, but not limited to, closed G containers. Once the tumor segment is added to the closed system, the system is closed to the outside environment until the TIL is ready to be administered to the patient.

As used herein, the terms "fragmenting," "fragment," and "fragmented" describe the process of destroying a tumor, including mechanical fragmentation methods such as crushing, slicing, dividing, and comminuting a tumor tissue, and any other method used to disrupt the physical structure of tumor tissue.

The terms "peripheral blood mononuclear cells" and "PBMC" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen presenting cells (PBMCs are one type of antigen presenting cells), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

The terms "peripheral blood lymphocytes" and "PBL" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from donors. In some embodiments, PBLs are isolated from whole blood or apheresis products from donors by positively or negatively selecting for a T cell phenotype, such as a CD3+CD45+ T cell phenotype.

The term "anti-CD3 antibody" refers to an antibody against the CD3 receptor in the T cell antigen receptor of mature T cells or a variant thereof, such as a monoclonal antibody, and includes human, humanized, chimeric, murine or mammalian animal antibodies. Anti-CD3 antibodies include OKT-3, also known as murozumab. Anti-CD3 antibodies also include UHCT1 clones, also known as T3 and CD3ε. Other anti-CD3 antibodies include, eg, otelixizumab, teplizuma, and visilizumab.

The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or a biosimilar or variant thereof against the CD3 receptor among the T cell antigen receptors of mature T cells, including human , humanized, chimeric or murine antibodies, and include commercially available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc , San Diego, California, USA Diego, CA, USA)) and murozumab or its variants, conservative amino acid substitutions, glycated forms or biosimilars. The amino acid sequences of the heavy and light chains of murozumab are given in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A fusionoma capable of producing OKT-3 is deposited with the American Type Culture Collection (American Type Culture Collection) and assigned ATCC accession number CRL 8001. A fusionoma capable of producing OKT-3 is also deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

The term "IL-2" (also referred to herein as "IL2") refers to the T-cell growth factor known as interleukin-2, and includes all forms of IL-2, including human and mammalian forms, the conserved amine Amino acid substitutions, glycated forms, biosimilars and their variants. IL-2 is described in, for example, Nelson, J. Immunol . 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol . 2008, 26, 453- 79, the disclosure of which is incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, available from several suppliers, 22 million IU per single-use vial) and the US-NH Recombinant forms of IL-2 supplied by CellGenix, Inc., Tsmouth, or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b) and other commercial equivalents from other suppliers . Aldesleukin (desalamidyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukins suitable for use in the present invention is given in Table 2 (SEQ ID NO: 4). The term IL-2 also encompasses pegylated forms of IL-2 as described herein, including the pegylated IL2 prodrug bempegaldesleukin (NKTR-214, as in SEQ ID NO:4 PEGylated human recombinant IL-2, in which an average of 6 lysine residues are modified by [(2,7-bis{[methylpoly(oxyethylene)]aminoformyl}-9H- 9-yl)methoxy]carbonyl substituted N ⁶ ), which can be purchased from Nektar Therapeutics, South San Francisco, California, USA, or can be prepared by methods known in the art, such as International Patent Application Publication No. WO 2018 /132496 A1, Example 19 or the method described in US Patent Application Publication No. US 2019/0275133 A1, Example 1, the disclosures of which are incorporated herein by reference. Bempegaldesleukin (NKTR-214) and other pegylated IL2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication No. US 2014/0328791 A1 and International Patent Application Publication No. WO 2012/ 065086 Al, the disclosure of which is incorporated herein by reference. Alternative forms of binding IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, which The disclosure is incorporated herein by reference. IL-2 formulations suitable for use in the present invention are described in US Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

離胺酸、烯丙氧基羰基離胺酸、2-胺基-8-側氧基壬酸、2-胺基-8-側氧基辛酸、對乙醯基-L-苯丙胺酸、對疊氮基甲基-L-苯丙胺酸(pAMF)、對碘-L-苯丙胺酸、間乙醯基苯丙胺酸、2-胺基-8-側氧基壬酸、對炔丙基氧基苯丙胺酸、對炔丙基-苯丙胺酸、3-甲基-苯丙胺酸、L-多巴(L-Dopa)、氟化苯丙胺酸、異丙基-L-苯丙胺酸、對疊氮基-L-苯丙胺酸、對醯基-L-苯丙胺酸、對苯甲醯基-L-苯丙胺酸、對溴苯基丙胺酸、對胺基-L-苯丙胺酸、異丙基-L-苯丙胺酸、O-烯丙基酪胺酸、O-甲基-L-酪胺酸、O-4-烯丙基-L-酪胺酸、4-丙基-L-酪胺酸、膦醯基酪胺酸、三-O-乙醯基-GlcNAcp-絲胺酸、L-磷絲胺酸、膦醯基絲胺酸、L-3-(2-萘基)丙胺酸、2-胺基-3-((2-((3-(苯甲氧基)-3-側氧基丙基)胺基)乙基)硒烷基)丙酸、2-胺基-3-(苯基硒烷基)丙酸或硒半胱胺酸。在一些實施例中，相對於野生型IL-2多肽，IL-2結合物與IL-2受體α(IL-2Rα)次單元之親和力降低。在一些實施例中，相對於野生型IL-2多肽，降低之親和力係與IL-2Rα之結合親和力降低約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、99%或大於99%。在一些實施例中，相對於野生型IL-2多肽，降低之親和力係約1倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、30倍、50倍、100倍、200倍、300倍、500倍、1000倍或更大。在一些實施例中，結合部分削弱或阻斷IL-2與IL-2Rα之結合。在一些實施例中，結合部分包含水溶性聚合物。在一些實施例中，另外的結合部分包含水溶性聚合物。在一些實施例中，水溶性聚合物各獨立地包含聚乙二醇(PEG)、聚(丙二醇)(PPG)、乙二醇及丙二醇之共聚物、聚(氧乙基化多元醇)、聚(烯醇)、聚(乙烯吡咯啶酮)、聚(羥烷基甲基丙烯醯胺)、聚(羥烷基甲基丙烯酸酯)、聚(醣)、聚(α-羥基酸)、聚(乙烯醇)、聚磷氮烯、聚

唑啉(POZ)、聚(N-丙烯醯嗎啉)或其組合。在一些實施例中，水溶性聚合物各獨立地包含PEG。在一些實施例中，PEG為線性PEG或分支鏈PEG。在一些實施例中，水溶性聚合物各獨立地包含多醣。在一些實施例中，多醣包含聚葡萄糖、聚唾液酸(PSA)、玻尿酸(HA)、直鏈澱粉、肝素、硫酸乙醯肝素(HS)、糊精或羥乙基澱粉(HES)。在一些實施例中，水溶性聚合物各獨立地包含聚醣。在一些實施例中，水溶性聚合物各獨立地包含多元胺。在一些實施例中，結合部分包含蛋白質。在一些實施例中，另外的結合部分包含蛋白質。在一些實施例中，蛋白質各獨立地包含白蛋白、轉鐵蛋白(transferrin)或運甲狀腺素蛋白(transthyretin)。在一些實施例中，蛋白質各獨立地包含Fc部分。在一些實施例中，蛋白質各獨立地包含IgG之Fc部分。在一些實施例中，結合部分包含多肽。在一些實施例中，另外的結合部分包含多肽。在一些實施例中，多肽各獨立地包含XTEN肽、富甘胺酸高胺基酸聚合物(HAP)、PAS多肽、彈性蛋白樣多肽(ELP)、CTP肽或明膠樣蛋白質(GLK)聚合物。在一些實施例中，分離及純化之IL-2多肽藉由麩胺醯化修飾。在一些實施例中，結合部分直接結合至分離及純化之IL-2多肽。在一些實施例中，結合部分經由連接子間接結合至分離及純化之IL-2多肽。在一些實施例中，連接子包含同型雙官能連接子。在一些實施例中，同型雙官能連接子包含羅曼特氏試劑(Lomant's reagent)二硫代雙(琥珀醯亞胺基丙酸酯)DSP、3'3'-二硫代雙(丙酸磺基琥珀醯亞胺酯)(DTSSP)、辛二酸二琥珀醯亞胺酯(DSS)、辛二酸雙(磺基琥珀醯亞胺酯)(BS)、酒石酸二琥珀醯亞胺酯(DST)、酒石酸二磺基琥珀醯亞胺酯(磺基DST)、糖基雙(琥珀醯亞胺基丁二酸)伸乙酯(EGS)、戊二酸二琥珀醯亞胺酯(DSG)、碳酸N,N'-二琥珀醯亞胺酯(DSC)、二亞胺代二酸二甲酯(DMA)、庚二亞胺酸二甲酯(DMP)、辛二亞胺酸二甲酯(DMS)、二甲基-3,3'-二硫代雙丙醯亞胺酸酯(DTBP)、1,4-二(3'-(2'-吡啶基二硫基)丙醯胺基)丁烷(DPDPB)、雙順丁烯二醯亞胺基己烷(BMH)、含有芳基鹵化物之化合物(DFDNB)(諸如1,5-二氟-2,4-二硝基苯或1,3-二氟-4,6-二硝基苯)、4,4'-二氟-3,3'-二硝基苯基碸(DFDNPS)、雙-[β-(4-疊氮基柳基醯胺基)乙基]二硫化物(BASED)、甲醛、戊二醛、1,4-丁二醇二縮水甘油醚、己二酸二醯肼、碳醯肼、鄰甲苯胺、3,3'-二甲基聯苯胺、聯苯胺、α,α'-對二胺基聯苯、二碘-對二甲苯磺酸、N,N'-伸乙基-雙(碘乙醯胺)或N,N'-六亞甲基-雙(碘乙醯胺)。在一些實施例中，連接子包含異型雙官能連接子。在一些實施例中，異型雙官能連接子包含3-(2-吡啶基二硫基)丙酸N-琥珀醯亞胺酯(sPDP)、長鏈3-(2-吡啶基二硫基)丙酸N-琥珀醯亞胺酯(LC-sPDP)、水溶性長鏈3-(2-吡啶基二硫基)丙酸N-琥珀醯亞胺酯(磺基-LC-sPDP)、琥珀醯亞胺基氧基羰基-α-甲基-α-(2-吡啶基二硫基)甲苯(sMPT)、磺基琥珀醯亞胺基-6--[α-甲基-α-(2-吡啶基二硫基)甲苯醯胺基]己酸酯(磺基-LC-sMPT)、琥珀醯亞胺基-4-(N-順丁烯二醯亞胺基甲基)環己烷-1-甲酸酯(sMCC)、磺基琥珀醯亞胺基-4-(N-順丁烯二醯亞胺基甲基)環己烷-1-甲酸酯(磺基-sMCC)、間順丁烯二醯亞胺基苯甲醯基-N-羥基琥珀醯亞胺酯(MBs)、間順丁烯二醯亞胺基苯甲醯基-N-羥基磺基琥珀醯亞胺酯(磺基-MBs)、(4-碘乙醯基)胺基苯甲酸N-琥珀醯亞胺酯(sIAB)、(4-碘乙醯基)胺基苯甲酸磺基琥珀醯亞胺酯(磺基-sIAB)、琥珀醯亞胺基-4-(對順丁烯二醯亞胺基苯基)丁酸酯(sMPB)、磺基琥珀醯亞胺基-4-(對順丁烯二醯亞胺基苯基)丁酸酯(磺基-sMPB)、N-(γ-順丁烯二醯亞胺基丁醯氧基)琥珀醯亞胺酯(GMBs)、N-(γ-順丁烯二醯亞胺基丁醯氧基)磺基琥珀醯亞胺酯(磺基-GMBs)、6-((碘乙醯基)胺基)己酸琥珀醯亞胺酯(sIAX)、6-[6-(((碘乙醯基)胺基)己醯基)胺基]己酸琥珀醯亞胺酯(sIAXX)、4-(((碘乙醯基)胺基)甲基)環己烷-1-甲酸琥珀醯亞胺酯(sIAC)、6-(((((4-碘乙醯基)胺基)甲基)環己烷-1-羰基)胺基)己酸琥珀醯亞胺酯(sIACX)、碘乙酸對硝苯酯(NPIA)、羰基反應性及硫氫基反應性交聯劑，諸如4-(4-N-順丁烯二醯亞胺基苯基)丁酸醯肼(MPBH)、4-(N-順丁烯二醯亞胺基甲基)環己烷-1-羧基-醯肼-8(M2C2H)、3-(2-吡啶基二硫基)丙醯基醯肼(PDPH)、N-羥基琥珀醯亞胺基-4-迭氮柳酸(NHs-AsA)、N-羥基磺基琥珀醯亞胺基-4-迭氮水楊酸(磺基-NHs-AsA)、磺基琥珀醯亞胺基-(4-迭氮柳基醯胺基己酸酯(磺基-NHs-LC-AsA)、磺基琥珀醯亞胺基-2-(對迭氮柳基醯胺基)乙基-1,3'-二硫丙酸酯(sAsD)、N-羥基琥珀醯亞胺基-4-迭氮苯甲酸酯(HsAB)、N-羥基磺基琥珀醯亞胺基-4-迭氮苯甲酸酯(磺基-HsAB)、N-琥珀醯亞胺基-6-(4'-疊氮基-2'-硝基苯基胺基)己酸酯(sANPAH)、磺基琥珀醯亞胺基-6-(4'-疊氮基-2'-硝基苯基胺基)己酸酯(磺基-sANPAH)、N-5-疊氮基-2-硝基苯甲醯氧基丁二醯亞胺(ANB-NOs)、磺基琥珀醯亞胺基-2-(間疊氮基-鄰硝基苯甲醯胺基)-乙基-1,3'-二硫丙酸酯(sAND)、N-琥珀醯亞胺基-4(4-迭氮苯基)1,3'-二硫丙酸酯(sADP)、(4-迭氮苯基)-1,3'-二硫丙酸N-磺基琥珀醯亞胺酯(磺基-sADP)、4-(對迭氮苯基)丁酸磺基琥珀醯亞胺酯(磺基-sAPB)、2-(7-疊氮基-4-甲基香豆素-3-乙醯胺)乙基-1,3'-二硫丙酸磺基琥珀醯亞胺酯(sAED)、7-疊氮基-4-甲基香豆素-3-乙酸磺基琥珀醯亞胺酯(磺基-sAMCA)、重氮丙酮酸對硝苯酯(ρNPDP)、對硝苯基-2-重氮-3,3,3-三氟丙酸酯(PNP-DTP)、1-(對疊氮基柳基醯胺基)-4-(碘乙醯胺基)丁烷(AsIB)、N-[4-(對疊氮基柳基醯胺基)丁基]-3'-(2'-吡啶基二硫基)丙醯胺(APDP)、二苯甲酮-4-碘乙醯胺、對疊氮基苯甲醯基醯肼(ABH)、4-(對疊氮基柳基醯胺基)丁胺(AsBA)或對迭氮苯基乙二醛(APG)。在一些實施例中，連接子包含可裂解連接子，視情況包含二肽連接子。在一些實施例中，二肽連接子包含Val-Cit、Phe-Lys、Val-Ala或Val-Lys。在一些實施例中，連接子包含不可裂解連接子。在一些實施例中，連接子包含順丁烯二醯亞胺基，視情況包含順丁烯二醯亞胺基己醯基(mc)、琥珀醯亞胺基-4-(N-順丁烯二醯亞胺基甲基)環己烷-1-甲酸酯(sMCC)或磺基琥珀醯亞胺基-4-(N-順丁烯二醯亞胺基甲基)環己烷-1-甲酸酯(磺基-sMCC)。在一些實施例中，連接子進一步包含間隔子。在一些實施例中，間隔子包含對胺基苯甲基醇(PAB)、對胺基苯甲氧基羰基(PABC)、其衍生物或類似物。在一些實施例中，結合部分能夠延長IL-2結合物之血清半衰期。在一些實施例中，另外的結合部分能夠延長IL-2結合物之血清半衰期。在一些實施例中，適用於本發明之IL-2形式為本文所描述之任一種IL-2形式的片段。在一些實施例中，適用於本發明之IL-2形式係如美國專利申請公開案US 2020/0181220 A1號及美國專利申請公開案US 2020/0330601 A1號中所揭示般聚乙二醇化。在一些實施例中，適用於本發明之IL-2形式為IL-2結合物，其包含：IL-2多肽，其包含N6-疊氮基乙氧基-離胺酸(AzK)，其共價連接至包含聚乙二醇(PEG)之結合部分，其中：IL-2多肽包含與SEQ ID NO: 5具有至少80%序列一致性之胺基酸序列；及參照SEQ ID NO: 5中的胺基酸位置對於位置K35、F42、F44、K43、E62、P65、R38、T41、E68、Y45、V69或L72處之胺基酸的AzK取代物。在一些實施例中，IL-2多肽包含相對於SEQ ID NO:5的一個殘基之N端缺失。在一些實施例中，適用於本發明之IL-2形式缺乏IL-2R α鏈接合，但保持與中間親和力IL-2R β-γ傳訊複合物的正常結合。 In some embodiments, a form of IL-2 suitable for use in the present invention is THOR-707 commercially available from Synthorx, Inc. The preparation and characterization of THOR-707 and other alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US 2020/0181220 A1 and US 2020/0330601 A1, the disclosures of which are incorporated by reference way incorporated into this article. In some embodiments, a form of IL-2 suitable for use in the present invention is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and at an amino acid selected from Binding moieties that bind to isolated and purified IL-2 polypeptides at positions: K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, where the amine The numbering of amino acid residues corresponds to SEQ ID NO:5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is at E62. In some embodiments, the amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to an isoamine acid, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to a non-natural amino acids. In some embodiments, the unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN- L-lysine, norbornene lysine, TCO-lysine, methyl tetra

Lysine, Allyloxycarbonyllysine, 2-Amino-8-Oxynonanoic Acid, 2-Amino-8-Oxylocaprylic Acid, p-Acetyl-L-Phenylalanine, Diagram Nitromethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxo-nonanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa (L-Dopa), fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl Tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O -Acetyl-GlcNAcp-serine, L-phosphoserine, phosphonylserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-( (3-(Benzyloxy)-3-oxopropyl)amino)ethyl)selenyl)propionic acid, 2-amino-3-(phenylselenyl)propionic acid or selenium semi cystine. In some embodiments, the IL-2 binder has reduced affinity for the IL-2 receptor alpha (IL-2Rα) subunit relative to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% reduced binding affinity for IL-2Rα relative to a wild-type IL-2 polypeptide %, 90%, 95%, 99% or greater than 99%. In some embodiments, the reduced affinity is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, relative to a wild-type IL-2 polypeptide. 30x, 50x, 100x, 200x, 300x, 500x, 1000x or more. In some embodiments, the binding moiety attenuates or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the binding moiety comprises a water soluble polymer. In some embodiments, the additional binding moiety comprises a water soluble polymer. In some embodiments, the water soluble polymers each independently comprise polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly (enol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(sugar), poly(alpha-hydroxyacid), poly (vinyl alcohol), polyphosphazene, poly

Oxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, each water soluble polymer independently comprises PEG. In some embodiments, the PEG is linear PEG or branched chain PEG. In some embodiments, each water soluble polymer independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises polydextrose, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each water soluble polymer independently comprises a polysaccharide. In some embodiments, each water-soluble polymer independently comprises a polyamine. In some embodiments, the binding moiety comprises a protein. In some embodiments, additional binding moieties comprise proteins. In some embodiments, each protein independently comprises albumin, transferrin, or transthyretin. In some embodiments, each protein independently comprises an Fc portion. In some embodiments, each protein independently comprises an Fc portion of IgG. In some embodiments, the binding moiety comprises a polypeptide. In some embodiments, additional binding moieties comprise polypeptides. In some embodiments, the polypeptides each independently comprise an XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer . In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the binding moiety binds directly to the isolated and purified IL-2 polypeptide. In some embodiments, the binding moiety is indirectly bound to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lomant's reagent dithiobis(succinimidylpropionate) DSP, 3'3'-dithiobis(sulfopropionate) Succinimidyl suberate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl suberate) (BS), disuccinimidyl tartrate (DST) , disuccinimidyl tartrate (sulfo-DST), glycosyl bis(succinimidylsuccinate) ethylidene (EGS), disuccinimidyl glutarate (DSG), carbonic acid N,N'-disuccinimidyl ester (DSC), dimethyl diiminodiacid (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS ), dimethyl-3,3'-dithiobisacryl imidate (DTBP), 1,4-bis(3'-(2'-pyridyldithio)acrylamide) butyl alkane (DPDPB), bismaleimidohexane (BMH), compounds containing aryl halides (DFDNB) (such as 1,5-difluoro-2,4-dinitrobenzene or 1, 3-difluoro-4,6-dinitrobenzene), 4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalpine Amino) ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, dihydrazide adipate, carbohydrazine, o-toluidine, 3, 3'-Dimethylbenzidine, benzidine, α,α'-p-diaminobiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylidene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker comprises N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long chain 3-(2-pyridyldithio)propane Acid N-succinimide ester (LC-sPDP), water-soluble long-chain 3-(2-pyridyldithio)propionic acid N-succinimide ester (sulfo-LC-sPDP), succinimide Aminooxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6--[α-methyl-α-(2-pyridine Dithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1- Formate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-cis-butyl Eniminobenzoyl-N-hydroxysuccinimidyl esters (MBs), maleimidobenzoyl-N-hydroxysulfosuccinimidyl esters (sulfo -MBs), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo- sIAB), succinimidyl-4-(p-maleimidephenyl) butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimide phenyl)butyrate (sulfo-sMPB), N-(γ-maleiminobutyryloxy)succinimidyl esters (GMBs), N-(γ-maleimido Aminobutyryloxy)sulfosuccinimidyl esters (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), 6-[6 -(((iodoacetyl)amino)hexyl)amino]hexanoic acid succinimidyl ester (sIAXX), 4-(((iodoacetyl)amino)methyl)cyclohexane- Succinimidyl 1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoic acid succinimidyl (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers such as 4-(4-N-maleiminophenyl)butyric acid hydrazide ( MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxy-hydrazide-8(M2C2H), 3-(2-pyridyldithio)propionyl-acyl Hydrazine (PDPH), N-hydroxysuccinimidyl-4-azosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azosalicylic acid (sulfo-NHs- AsA), sulfosuccinimidyl-(4-azosylsylamidohexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azosylsylamido N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinyl Imino-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimido-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido- 2-Nitrobenzoyloxybutadiimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1 ,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), (4-azidophenyl base)-1,3'-dithiopropionic acid N-sulfosuccinimidyl ester (sulfo-sADP), 4-(p-azidophenyl)butyric acid sulfosuccinimidyl ester (sulfo- sAPB), 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionic acid sulfosuccinimidyl ester (sAED), 7 -Azido-4-methylcoumarin-3-sulfosuccinimidyl acetate (sulfo-sAMCA), p-nitrophenyldiazopyruvate (ρNPDP), p-nitrophenyl-2-heavy Nitrogen-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N- [4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-Azidobenzoylhydrazine (ABH), 4-(p-Azidosalicylamido)butylamine (AsBA) or p-Azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises maleimide, optionally maleimidocaproyl (mc), succinimidyl-4-(N-male Diimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1 -Formate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), derivatives or analogs thereof. In some embodiments, the binding moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional binding moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, a form of IL-2 suitable for use in the present invention is a fragment of any of the forms of IL-2 described herein. In some embodiments, forms of IL-2 suitable for use in the invention are pegylated as disclosed in US Patent Application Publication No. US 2020/0181220 Al and US Patent Application Publication No. US 2020/0330601 Al. In some embodiments, a form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-lysine (AzK) co- is linked to a binding moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 5; and with reference to SEQ ID NO: 5 Amino acid positions AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of one residue of SEQ ID NO:5. In some embodiments, forms of IL-2 suitable for use in the invention lack IL-2R alpha chain binding, but retain normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.

In some embodiments, a form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-lysine (AzK) co- is linked to a binding moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5; and with reference to the amino acid sequence in SEQ ID NO:5 Amino acid positions AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72. In some embodiments, a form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-lysine (AzK) co- is linked to a binding moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:5; and with reference to the amino acid sequence in SEQ ID NO:5 Amino acid positions AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72. In some embodiments, a form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-lysine (AzK) co- is linked to a binding moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence with at least 98% sequence identity to SEQ ID NO:5; and with reference to the amino acid sequence in SEQ ID NO:5 Amino acid positions AzK substitutions for amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72.

In some embodiments, the IL-2 form nemvaleukin alfa (also known as ALKS-4230 (SEQ ID NO: 6) suitable for use in the present invention is available from Alkermes , Inc.)). Inner interleukin α is also known as human ^{interleukin 2} fragment (1-59) variant (Cys ¹²⁵ >Ser ⁵¹ ), which is fused to human interleukin 2 fragment ( 62-132), fused to the human interleukin-2 receptor alpha chain fragment (139-303) via a peptidyl linker ( ¹³³ GSGGGS ¹³⁸ ), produced in Chinese hamster ovary (CHO) cells, glycosylated; human Interleukin 2 (IL-2) (75-133)-peptide [[Cys ¹²⁵ (51)>Ser]-mutant variant (1-59) fused to human interleukin via G2 peptide linker (60-61) Interleukin 2 (IL-2) (4-74)-peptide (62-132) and fused to _human interleukin 2 receptor α chain (IL2R subunit α, IL2R subunit α, IL2Rα, IL2RA) (1-165)-peptide (139-303), produced in Chinese hamster ovary (CHO) cells, in the glycosylated form alpha. The amino acid sequence of interleukin alpha is provided in SEQ ID NO:6. In some embodiments, interleukin alpha exhibits the following post-translational modifications: disulfide bonds at positions: 31-116, 141-285, 184-242, 269-301, 166-197, or 166-199, 168-199 or 168-197 (using the numbering in SEQ ID NO:6), and the glycosylation sites at positions N187, N206, T212 using the numbering in SEQ ID NO:6. The preparation and characterization of interleukin alfa and other alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication No. US 2021/0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated by reference way incorporated into this article. In some embodiments, a form of IL-2 suitable for use in the invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, forms of IL-2 suitable for use in the invention have the amino acid sequence set forth in SEQ ID NO: 6 or conservative amino acid substitutions thereof. In some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO: 7, or a variant, fragment or derivative thereof. In some embodiments, a form of IL-2 suitable for use in the present invention comprises at least 80%, at least 90%, at least 95% of amino acids 24-452 of SEQ ID NO: 7, or variants, fragments, or derivatives thereof. A fusion protein of an amino acid sequence with % or at least 90% sequence identity. Other forms of IL-2 suitable for use in the present invention are described in US Patent No. 10,183,979, the disclosure of which is incorporated herein by reference. Optionally, in some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner via a mucin domain polypeptide linker , wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity with IL-1Rα and having receptor antagonist activity of IL-Rα, and wherein the second fusion partner comprises An immunoglobulin comprising an Fc region in whole or in part, wherein the mucin domain polypeptide linker comprises SEQ ID NO: 8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8, and wherein the fusion protein The half-life is improved compared to fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker.

In some embodiments, forms of IL-2 suitable for use in the present invention include antibody interleukin graft proteins comprising: heavy chain variable regions (VH) comprising complementarity determining regions HCDR1, HCDR2, HCDR3; light chain variable region (VL), which comprises LCDR1, LCDR2, LCDR3; and IL-2 molecule or fragment thereof, which is grafted into the CDR of VH or VL, wherein the antibody cytokine graft protein is preferential to the regulatory T cells expand T effector cells. In some embodiments, the antibody cytokine graft protein comprises a heavy chain variable region (V _H ), which comprises complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (V _L ), which comprises LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into a CDR of a _VH or _VL , wherein the IL-2 molecule is a mutein, and wherein the antibody interleukin graft protein preferentially expands over regulatory T cells T effector cells. In some embodiments, the IL-2 regimen comprises administering an antibody described in US Patent Application Publication No. US 2020/0270334 Al, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine graft protein comprises: a heavy chain variable region (VH) comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL) comprising LCDR1, LCDR2, LCDR3 and an IL-2 molecule or fragment thereof, which is grafted into the CDR of _VH or _VL ; wherein the IL-2 molecule is a mutant protein (mutein), wherein the antibody interleukin graft protein is relative to the regulatory T cell T effector cells are preferentially expanded, and wherein the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of an IgG class light chain comprising SEQ ID NO: 39 and an IgG class comprising SEQ ID NO: 38 IgG class heavy chain; IgG class light chain comprising SEQ ID NO: 37 and IgG class heavy chain comprising SEQ ID NO: 29; IgG class light chain comprising SEQ ID NO: 39 and IgG class comprising SEQ ID NO: 29 heavy chain; and an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 38.

In some embodiments, an IL-2 molecule or a fragment thereof is grafted into HCDR1 of the _VH , wherein the IL-2 molecule is a mutein. In some embodiments, the IL-2 molecule or fragment thereof is grafted into the HCDR2 of the _VH , wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is grafted into HCDR3 of the _VH , wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is grafted into LCDR1 of the _VL , wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is grafted into LCDR2 of the _VL , wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is grafted into LCDR3 of the _VL , wherein the IL-2 molecule is a mutein.

Insertion of the IL-2 molecule can be at or near the N-terminal region of the CDRs, in the middle region of the CDRs, or at or near the C-terminal region of the CDRs. In some embodiments, the antibody interleukin graft protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL2 sequence does not frame the CDR sequence. In some embodiments, the antibody interleukin graft protein comprises an IL-2 molecule incorporated into the CDRs, wherein the IL-2 sequence replaces all or a portion of the CDR sequence. The IL-2 molecular replacement can be at the N-terminal region of the CDRs, in the middle region of the CDRs, or at or near the C-terminal region of the CDRs. IL-2 molecular substitutions can be as little as one or two amino acids in the CDR sequence or the entire CDR sequence.

In some embodiments, the IL-2 molecule is grafted directly into the CDR without a peptide linker, wherein there are no additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is grafted indirectly into a CDR with a peptide linker, wherein there are one or more additional amino acids between the CDR sequence and the IL-2 sequence.

In some embodiments, the IL-2 molecules described herein are IL-2 muteins. In some instances, the IL-2 muteins comprise a R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, the IL-2 mutein comprises the amino acid sequence in Table 1 of US Patent Application Publication No. US 2020/0270334 A1, the disclosure of which is incorporated herein by reference.

In some embodiments, the antibody interleukin graft protein comprises HCDR1 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25. In some embodiments, the antibody interleukin graft protein comprises HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16. In some embodiments, the antibody interleukin graft protein comprises HCDR1 selected from the group consisting of: selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 26 HCDR2. In some embodiments, the antibody interleukin graft protein comprises HCDR3 selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 27. In some embodiments, the antibody interleukin graft protein comprises a _VH region comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody interleukin graft protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody interleukin graft protein comprises a _VL region comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody interleukin graft protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody interleukin graft protein comprises a _VH region comprising the amino acid sequence of SEQ ID NO: 28; and a _VL region comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody interleukin graft protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 29; and a light chain region comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody interleukin graft protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 29; and a light chain region comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody interleukin graft protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 38; and a light chain region comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody interleukin graft protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 38; and a light chain region comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody interleukin graft protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020/0270334 A1 or variants, derivatives or fragments thereof, or conserved Amino acid substitutions, or proteins with at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody component of the antibody interleukin graft protein described herein comprises the immunoglobulin sequence, framework sequence or CDR sequence of palivizumab. In some embodiments, the antibody interleukin graft protein described herein has a longer serum half-life than a wild-type IL-2 molecule such as but not limited to aldesleukin or a comparable molecule. In some embodiments, the antibody interleukin graft protein described herein has a sequence as set forth in Table 3.

The term "IL-4" (also referred to herein as "IL4") refers to an interleukin known as interleukin 4, which is produced by Th2 T cells and eosinophils, basophils and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. After activation by IL-4, Th2 T cells then produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and MHC class II expression, and induces class switching from B cells to IgE and IgGl expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from a number of suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, NJ, USA (Catalogue # CYT-211 ) and Cyprus Inc. of Waltham, MA, USA. ThermoFisher Scientific, Inc. (Human IL-15 Recombinant Protein, Cat. No. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 9).

The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin 7, which can be obtained from stromal and epithelial cells as well as dendritic cells. Fry and Mackall , " Blood " 2002 , 99 , 3892-904 . IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer composed of IL-7 receptor alpha and a common gamma chain receptor, which is essential for T cell development in the thymus and survival in the periphery. A series of important signals. Recombinant human IL-7 suitable for use in the present invention is commercially available from a number of suppliers including ProSpec-Tany TechnoGene Ltd. of East Brunswick, NJ, USA (Catalogue No. CYT-254) and Cyprus of Waltham, MA, USA. Mo Fisher Scientific (Human IL-15 Recombinant Protein, Cat. No. Gibco PHC0071 ). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 10).

The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor known as interleukin-15, and includes all forms of IL-2, including human and mammalian forms, the conserved amine Amino acid substitutions, glycated forms, biosimilars and their variants. IL-15 is described, eg, in Fehniger and Caligiuri, Blood 2001 , 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 and IL-2 share β and γ signaling receptor subunits. Recombinant human IL-15 is a single non-glycosylated polypeptide chain containing 114 amino acids (and N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and Thermo Fisher Scientific, Waltham, MA, USA Science, Inc. (Human IL-15 Recombinant Protein, Cat. No. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 11).

The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic interleukin protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, Conservative amino acid substitutions, glycated forms, biosimilars and their variants. IL-21 is described, eg, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is mainly produced by natural killer T cells and activated human CD4 ⁺ T cells. Recombinant human IL-21 is a single non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-408-b) and Thermo Fisher Scientific, Waltham, MA, USA. Science, Inc. (Human IL-21 Recombinant Protein, Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 21).

When an "antitumor effective amount", "tumor inhibitory effective amount" or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by the physician taking into account the age, body weight, tumor size, infection or metastasis of the patient (individual) Individual differences in degree and symptoms are determined. It can generally be stated that the pharmaceutical compositions described herein comprising tumor infiltrating lymphocytes (e.g. passaged TILs or genetically modified cytotoxic lymphocytes) can contain 10 ⁴ to 10 ¹¹ cells/kg body weight (e.g. 10 ⁵ to 10 ⁶ , 10 ⁵ to 10 ¹⁰ , 10 ⁵ to 10 ¹¹ , 10 ⁶ to 10 ¹⁰ , 10 ⁶ to 10 ¹¹ , 10 ⁷ to 10 ¹¹ , 10 ⁷ to 10 ¹⁰ , 10 ⁸ to 10 ¹¹ , 10 ⁸ to 10 ¹⁰ , 10 ⁹ to 10 ¹¹ or 10 ⁹ to 10 ¹⁰ cells/kg body weight), including all integer values within those ranges. TIL (including, in some cases, genetically engineered cytotoxic lymphocytes) compositions can also be administered in multiples of these doses. TILs (including, in some cases, genetically engineered TILs) can be administered using infusion techniques generally known in immunotherapy (see eg Rosenberg et al., New England Journal of Medicine 1988 , 319, 1676). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient's disease symptoms and adjusting treatment accordingly.

The term "hematological malignancy (hematologic malignancy)" or related terms refers to cancers and tumors of mammalian hematopoietic and lymphoid tissues (including but not limited to blood, bone marrow, lymph nodes, and tissues of the lymphatic system). Hematological malignancies are also known as "liquid tumors". Hematological malignancies include (but are not limited to) acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute mononuclear leukemia (AMoL), Hodgkin's lymphoma (Hodgkin's lymphoma) and non-Hodgkin's lymphoma. The term "B-cell hematologic malignancies" refers to hematologic malignancies affecting B-cells.

The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in the peripheral blood, may also be referred to herein as PBLs. The terms MIL, TIL and PBL are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

As used herein, the term "microenvironment" may refer to the solid or hematological tumor microenvironment as a whole or may refer to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to the complex mixture that "promotes neoplastic transformation, supports tumor growth and invasion, protects tumors from host immunity, encourages resistance to therapy, and provides an ecological niche in which dominant metastases thrive." (niche) cells, soluble factors, signaling molecules, extracellular matrix, and mechanical signaling", as described in Swartz et al., " Cancer Res. ", 2012 , 72 , 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare due to immunosuppression of the microenvironment.

In some embodiments, the invention includes a method of treating cancer with a population of TILs, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, a population of TILs can be provided wherein the patients are pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, nonmyeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m2/d for 5 days (Days 27 to 23 prior to TIL infusion). In some embodiments, following nonmyeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU/kg every 8 hours to achieve physiological tolerance .

Experimental findings suggest that lymphocyte depletion plays a key role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for elements of the immune system ("cytokine repertoire") prior to adoptive transfer of tumor-specific T lymphocytes. Accordingly, some embodiments of the invention employ a lymphocyte depletion step (sometimes also referred to as "immunosuppressive conditioning") in the patient prior to the introduction of the TILs of the invention.

The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds as described herein sufficient to achieve the intended use, including but not limited to the treatment of disease. A therapeutically effective amount may vary depending on the intended application (in vitro or in vivo) or the individual and disease condition being treated (eg, the weight, age and sex of the individual), the severity of the disease condition or the mode of administration. The term also applies to doses that will induce a specific response in target cells, such as decreased platelet adhesion and/or cell migration. The particular dosage will vary depending on the particular compound selected, the dosing regimen followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

The terms "treatment/treating/treat" and similar terms refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms, and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects attributable to the disease. As used herein, "treatment" encompasses any treatment of disease in mammals, especially humans, and includes: (a) preventing the disease in individuals who may be predisposed to it but have not been diagnosed with the disease; (b) Inhibiting a disease means arresting its development or progression; and (c) ameliorating a disease means regressing the disease and/or alleviating one or more symptoms of the disease. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition that elicits an immune response or confers immunity in the absence of a disease condition, such as in the case of a vaccine.

When used with reference to a portion of a nucleic acid or protein, the term "heterologous" indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are often recombinantly produced having two or more sequences from unrelated genes arranged to produce a new functional nucleic acid sequence, such as a promoter from one source and a coding sequence from another regions or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (eg, a fusion protein).

The terms "sequence identity", "percent identity" and "sequence percent identity" (or their synonyms) in the context of two or more nucleic acids or polypeptides , such as "99% identity") means that two or more sequences or subsequences are compared and aligned (introducing spaces where necessary) for maximum correspondence and do not consider any conservative amino acid substitutions as sequence identity When a sexual portion is present, the two or more sequences or subsequences are identical or have a specified percentage of nucleotide or amino acid residues identical. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the U.S. Government's National Center for Biotechnology Information BLAST website. A comparison between two sequences can be made using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, CA, USA), or MegAlign (commercially available from DNASTAR) are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine the appropriate parameters for maximal alignment with specific alignment software. In some embodiments, the preset parameters of the comparison software are used.

As used herein, the term "variant" encompasses, but is not limited to, antibodies or fusion proteins comprising an amino acid sequence that differs from, within the amino acid sequence of a reference antibody One or more substitutions, deletions and/or additions at or adjacent to certain positions. A variant may comprise one or more conservative substitutions in its amino acid sequence compared to that of a reference antibody. Conservative substitutions may involve, for example, substitutions of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins.

"Tumor infiltrating lymphocytes" or "TILs" herein means a population of cells initially obtained as white blood cells that have left an individual's bloodstream and migrated into tumors. TILs include, but are not limited to, CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and secondary TILs. "Primary TILs" are cells obtained (sometimes referred to as "freshly collected") from a patient tissue sample as outlined herein, and "secondary TILs" are any population of TIL cells that has been expanded or proliferated as discussed herein, Including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, a second expansion of TILs or a second additional expansion of TILs (such as the TILs described in step D of Figure 8, including TILs referred to as reREP TILs).

TILs can generally be defined biochemically (using cell surface markers) or functionally (by their ability to infiltrate tumors and effect therapy). TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors after reintroduction into a patient. TIL can be further characterized by potency—for example, if, for example, interferon (IFN) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL, TIL can be regarded as for potent. If, for example, the amount of interferon (IFNγ) released is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL, greater than about 300 pg/mL, greater than about 400 pg/mL , greater than about 500 pg/mL, greater than about 600 pg/mL, greater than about 700 pg/mL, greater than about 800 pg/mL, greater than about 900 pg/mL, greater than about 1000 pg/mL, TIL can be considered strong effective.

The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include altering the sugar moiety, the base moiety and/or the linkage between individual deoxyribonucleotides in the oligonucleotide.

The term "RNA" defines a molecule comprising at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of the b-D-ribofuranose moiety. The term RNA includes double-stranded RNA; single-stranded RNA; isolated RNA, such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA; An altered RNA that differs from naturally occurring RNA by one or more nucleotides. Nucleotides in the RNA molecules described herein may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. Such altered RNAs may be referred to as analogs or analogs of naturally occurring RNAs.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Unless any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic composition of the invention is contemplated. Additional active pharmaceutical ingredients such as other drugs may also be incorporated into the described compositions and methods.

The term "about" or "approximately" means within a statistically meaningful range of values. This range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5%. The permissible variance encompassed by the term "about" or "approximately" depends on the particular system under study and is readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not exact and need not be exact, but can be approximated as necessary and and/or greater or lesser, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those skilled in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is "about" or "approximately" whether or not so expressly stated. It should be noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

When used in original and modified form in the appended claims, the transitional terms "comprising", "consisting essentially of" and "consisting of" are used relative to which Additional claim elements or steps not described (if any) are excluded from the scope of the claimed claims to define the scope of the claims. The term "comprising" is intended to be inclusive or open-ended, and does not exclude any additional, non-recited elements, methods, steps or materials. The term "consisting of" does not include any element, step or material other than that specified in the claim, and in the latter case excludes impurities normally associated with the specified material. The term "consisting essentially of" limits the scope of a claim to the named elements, steps or materials and those elements, steps or materials that do not materially affect the basic and novel characteristics of the claimed invention. In alternative embodiments, all compositions, methods and kits described herein embodying the invention may be more specifically defined by any transitional terms "comprising," "consisting essentially of," and "consisting of."

The term "antibody" and its plural form "antibodies" refer to intact immunoglobulins and to any antigen-binding fragment ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as _VH ) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as _VL ) and a light chain constant region. The light chain constant region comprises one domain, _CL . The _VH and _VL regions of antibodies can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs) or hypervariable regions (HVRs), and which can be interspersed with more conserved regions, called framework regions ( FR). Each _VH and _VL is composed of three CDRs and four FRs arranged in the following order from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more epitopes. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule capable of being bound by an antibody or TCR if presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. In addition, antigens can be recognized by the immune system. In some embodiments, the antigen is capable of inducing a humoral immune response or a cellular immune response, leading to activation of B lymphocytes and/or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a Th cell epitope. An antigen may also have one or more epitopes (eg, a B epitope and a T epitope). In some embodiments, an antigen will preferably typically react with its corresponding antibody or TCR in a highly specific and selective manner, and not react with a variety of other antibodies or TCRs that may be induced by other antigens.

The terms "monoclonal antibody", "mAb", "monoclonal antibody composition" or plurals thereof refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for certain receptors can be produced using knowledge and techniques in the art by injecting test individuals with the appropriate antigen and then isolating fusionomas expressing antibodies with the desired sequence or functional characteristics. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibody). Fusoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into a host cell that does not otherwise produce the antibody protein (such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to achieve monoclonal antibody synthesis in recombinant host cells. Recombinant production of antibodies is described in more detail below.

As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more antibodies that retain the ability to specifically bind to an antigen fragment. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, i.e. monovalent fragments consisting of VL, _VH , _CL and CH1 domains; ₍ ii) F(ab') 2 fragments, that is, a bivalent fragment, which includes two Fab fragments connected by a disulfide bridge at the hinge region; (iii) an Fd fragment composed of _VH and CH1 domains; (iv) a V fragment composed of a single arm of an antibody Fv fragments composed of _L and V _H domains; (v) domain antibody (dAb) fragments (Ward et al., "Nature ( Nature ), 1989 , 341 , 544-546), which can be composed of a V _H or a V _L domain composition; and (vi) isolated complementarity determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows the formation of a single protein chain in which The _VL and _VH regions pair to form a monovalent molecule called a single-chain Fv (scFv); see for example Bird et al., Science 1988, 242, 423-426; and Huston et al., National Academy of Sciences Proceedings 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for use in the same manner as whole antibodies. In some embodiments, the scFv protein domain comprises a _VH portion and a _VL portion. If the V _L domain is the N-terminal part of the scFv molecule, the scFv molecule is expressed as V _L -LV _H , or if the V _H domain is the N-terminal part of the scFv molecule, it is expressed as V _H -LV _L. Used to make scFv Molecules and methods for designing suitable peptide linkers are described in U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; R. Raag and M. Whitlow, "Single Chain Fv (Single Chain Fvs.)" Joint American Society for Experimental Biology FASEB, Vol. 9:73-80 (1995); and RE Bird and BW Walker, "Single Chain Antibody Variable Regions", Trends in Biotechnology (TIBTECH), vol. 9: 132-137 (1991), the disclosure of which is incorporated herein by reference.

As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations induced by random or site-directed mutagenesis in vitro or introduced by somatic mutation in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "human monoclonal antibody" refers to an antibody displaying a single binding specificity and having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibody is produced by a fusion tumor comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) fused with an immortalized cell, which includes human Genomes of heavy chain transgene and light chain transgene.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) derived from transgenes for human immunoglobulin genes or Antibodies isolated from transchromosomally transgenic animals (such as mice) or fusion tumors made therefrom (further described below); (b) antibodies isolated from host cells transformed to express human antibodies, such as from transfectomas; ( c) antibodies isolated from recombinant, combinatorial human antibody repertoires; and (d) antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may undergo in vitro mutagenesis (or when using transgenic animals of human Ig sequences, in vivo somatic mutagenesis), and thus the _VH and V of the recombinant antibody The amino acid sequences of _{the L} regions are sequences that, while derived from and related to human germline _VH and _VL sequences, may not naturally occur in vivo within the germline repertoire of human antibodies.

As used herein, "isotype" refers to the antibody class (eg, IgM or IgGl) encoded by the heavy chain constant region genes.

The phrases "antibody that recognizes an antigen" and "antibody that has specificity for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

The term "human antibody derivative" refers to any modified form of a human antibody, including a conjugate of the antibody with another active pharmaceutical ingredient or antibody. The term "conjugate", "antibody-drug conjugate", "ADC" or "immunoconjugate" refers to an antibody or fragment thereof that binds to another therapeutic moiety, which can be used with other therapeutic moieties using methods available in the art. The antibodies described herein bind.

The terms "humanized antibody (humanized antibodies)" and "humanized" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. "Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are derived from residues from the hypervariable regions of the recipient by modifying them from a non-human species (donor antibody) with the desired specificity, affinity and capacity, such as mouse, rat, rabbit or Human immunoglobulin (recipient antibody) with 15 residues in the hypervariable region of non-human primates substituted. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further optimize antibody potency. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all hypervariable loops corresponding to those regions of a non-human immunoglobulin and all or substantially all FR regions are those regions of human immunoglobulin sequences. A humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For additional details, see Jones et al., Nature 1986, 321 , 522-525; Riechmann et al., Nature 1988, 332 , 323-329; and Presta, New Insights in Structural Biology ( Curr. Op.Struct .Biol. )》 1992 , 2, 593-596. The antibodies described herein can also be modified to employ any Fc variant known to confer effector function and/or improved (eg, decreased) FcR binding. Fc variants may include, for example, any of the amino acid substitutions disclosed in International Patent Application Publication Nos. WO 1988/07089 A1 , WO 1996/14339 A1 , WO 1998/05787 A1 , WO 1998 /23289 A1, WO 1999/51642 A1, WO 99/58572 A1, WO 2000/09560 A2, WO 2000/32767 A1, WO 2000/42072 A2, WO 2002/44215 A2, WO 2002/ 060919 A2, WO 2003/074569 A2, WO 2004/016750 A2, WO 2004/029207 A2, WO 2004/035752 A2, WO 2004/063351 A2, WO 2004/074455 A2, WO 2004/099249 A2, WO 2005/040217 A2, WO 2005/070963 A1, WO 2005/077981 A2, WO 2005/092925 A2, WO 2005/123780 A2, WO 2006/019447 A1, WO 2006/047350 A2 and WO 2006/085967 A2; and US Patent Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784; the disclosures of which are incorporated herein by reference.

The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

"Biabodies" are small antibody fragments that have two antigen-combining sites. A fragment includes a heavy chain variable domain ₍ VH) linked to a light chain variable domain ( _VL ) in the same polypeptide chain ( _VH - _VL or _VL - _VH ). By using a linker that is too short for pairing between two variable domains on the same chain, the variable domains are forced to pair with the complementary domains of another chain and two antigen binding sites are created. Diabodies are more fully described in, for example, European Patent No. EP 404,097; International Patent Publication No. WO 93/11161; and Bolliger et al., Proceedings of the National Academy of Sciences USA 1993 , 90 , 6444-6448.

The term "glycosylation" refers to modified derivatives of antibodies. Aglycosylated antibodies lack glycosylation. Glycosylation can be altered, for example, to increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. As described in US Patent Nos. 5,714,350 and 6,350,861, deglycosylation increases the affinity of the antibody for antigen. Additionally or alternatively, antibodies can be prepared with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisected GlcNAc structures. Such altered glycosylation patterns were shown to increase antibody potency. Such carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells for expressing recombinant antibodies of the invention to thereby produce antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6) fucosyltransferase), so that antibodies expressed in Ms704, Ms705, and Ms709 cell lines are in their Lack of fucose on carbohydrates. Ms704, Ms705, and Ms709 FUT8−/− cell lines were generated by targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004/0110704 or Yamane-Ohnuki et al. , "Biotechnology and Bioengineering ( Biotechnol. Bioeng. ), 2004 , 8 7, 614-622). As another example, European Patent No. EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines are due to reduced or eliminated α1,6 bond-related enzymes while exhibiting hypofucosylation, and also describe cell lines with lower enzymatic activity for adding fucose to N-acetylglucosamine bound to the Fc region of an antibody Or do not have the enzyme activity, such as rat myeloma cell line YB2/0 (ATCC CRL 1662). International Patent Publication WO 03/035835 describes a variant CHO cell line, Lec 13 cells, which has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in expression in that host cell Hypofucosylation of antibodies (see also Shields et al., J. Biol. Chem. 2002, 277 , 26733-26740. International Patent Publication WO 99/54342 describes engineering to Cell lines expressing glycoprotein-modified glycosyltransferases, such as β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), allow the expression of antibodies expressed in engineered cell lines Increased bipartite GlcNac structure, thereby improving the ADCC activity of the antibody (see also Umana et al., "Nat. Biotech ( Nat. Biotech. )" 1999, 17 , 176-180). Alternatively, the fucose residue of the antibody can be Cleavage using fucosidase. For example, fucosidase α-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino et al., "Biochemistry ( Biochem. )" 1975 , 14 , 5516-5523 as described.

"Pegylated" refers to a modified antibody or fragment thereof, typically reactive with polyethylene glycol (PEG), such as PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment ester or aldehyde derivatives. For example, pegylation can increase the biological (eg, serum) half-life of the antibody. Preferably, PEGylation is performed via acylation or alkylation with reactive PEG molecules (or similar reactive water-soluble polymers). As used herein, the term "polyethylene glycol" is intended to cover any form of PEG that has been used to derivatize other proteins, such as mono(C ₁ -C ₁₀ )alkoxy or aryloxy-polyethylene glycol or polyethylene glycol. Diol-maleimide. Antibodies to be pegylated may be deglycosylated antibodies. Pegylation methods are known in the art and can be applied to the antibodies of the present invention, as described, for example, in European Patent Nos. EP 0154316 and EP 0401384 and U.S. Patent No. 5,824,778, each of The disclosure is incorporated herein by reference.

The term "biosimilar" means a biological product, including a monoclonal antibody or protein, which is closely similar to a reference biological product approved in the United States despite minor differences in its non-clinically active components, and the biological product is closely related to the reference product. There were no clinically meaningful differences in the safety, purity and potency of the products. In addition, an analogous biological or "biosimilar" drug is a biological drug that is similar to another biological drug that has been licensed for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions. A biological product or biopharmaceutical is a drug made or derived from a biological source such as bacteria or yeast. It may consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), then the drug regulatory agency refers to aldesleukin to approve the protein "biologically similar" to aldesleukin or to be "proleukin" of aldesleukin biosimilars". In Europe, an analogous biological or "biosimilar" drug is a biological drug that is similar to another biological drug that has been licensed for use by the European Medicines Agency. The relevant legal basis for the application of biosimilars in Europe is Article 6 of Regulation (EC) No. 726/2004 and Article 10(4) of Directive 2001/83/EC as amended, and therefore in Europe, biosimilars can be approved under Regulation ( Article 6 of EC) No 726/2004 and Article 10(4) of Directive 2001/83/EC authorize, grant authorization or be the object of an application for authorization. Authorized original biomedical products can be called "reference medicinal products" in Europe. Some requirements for products considered as biosimilars are outlined in the CHMP Guidance on Similar Biological Medicinal Products. In addition, product-specific guidance, including guidance on monoclonal antibody biosimilars, will be provided by EMA on a product-by-product basis and published on its website. As described herein, a biosimilar may be similar to a reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and/or efficacy. Additionally, a biosimilar may be used or intended to be used in the treatment of the same condition as the reference medicinal product. Accordingly, a biosimilar as described herein can be considered to have similar or highly similar quality characteristics to the reference medicinal product. Alternatively or additionally, a biosimilar as described herein may be considered to have similar or highly similar biological activity to the reference medicinal product. Alternatively or additionally, a biosimilar as described herein may be considered to have a similar or highly similar safety profile to the reference medicinal product. Alternatively or additionally, a biosimilar as described herein may be considered to have similar or highly similar efficacy to a reference medicinal product. As described herein, European biosimilars were compared to reference medicinal products that had been licensed by EMA. However, in some cases biosimilars may be compared in certain studies with biopharmaceutical products licensed outside the EEA (non-EEA licensed "comparators"). Such studies include, for example, certain clinical and in vivo non-clinical studies. As used herein, the term "biosimilar" also relates to a biopharmaceutical product that has been or can be compared with a non-EEA licensed comparator. Certain biosimilars are proteins, such as antibodies, antibody fragments (eg, antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications in the amino acid structure, including, for example, deletions, additions and/or substitutions of amino acids that do not significantly affect the function of the polypeptide. A biosimilar may comprise an amino acid sequence that has 97% or greater (eg, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of its reference medicinal product. A biosimilar may contain one or more post-translational modifications, such as but not limited to, glycosylation, oxidation, desamidation, and/or truncation, that differ from the post-translational modification of the reference medicinal product, as long as the difference does not cause the medicinal product to Changes in safety and/or efficacy are sufficient. Biosimilars may have the same or different glycosylation patterns as the reference medicinal product. In particular, although not exclusively, biosimilars may have different glycosylation patterns if the differences address or are intended to address safety concerns associated with the reference medicinal product. In addition, a biosimilar may differ from a reference medicinal product in, for example, its strength, pharmaceutical form, formulation, excipients and/or presentation, as long as the safety and efficacy of the medicinal product are not affected. Biosimilars may contain differences, for example, in pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles compared to a reference medicinal product, but are still considered sufficiently similar to the reference medicinal product to be licensed or deemed as appropriate for the license. In some cases, biosimilars exhibit different binding characteristics than the reference medicinal product, where such different binding characteristics are not considered by regulatory authorities (such as the EMA) to be an obstacle to the licensing of similar biological products. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions. II. Gene Editing Process A. Overview: TIL Amplification + Gene Editing

Some embodiments of the invention relate to methods for expanding TIL populations (e.g., TIL populations enriched for CD39/CD69 negative and/or CD39 ^LO /CD69 ^LO ) comprising editing at least a portion of the TILs with one or more genes to enhance The steps of its therapeutic effect. As used herein, "gene-editing/gene editing" and "genome editing" refer to a genetic modification in which DNA is permanently modified in the genome of a cell, such as insertions, Deletion, modification or substitution of DNA. In some embodiments, gene editing results in the silencing (sometimes called gene knockout) or suppression/reduction (sometimes called gene attenuation) of the expression of a DNA sequence. In other embodiments, gene editing results in enhanced expression of the DNA sequence (eg, by causing overexpression). According to an embodiment of the present invention, gene editing technology is used to enhance the effectiveness of the therapeutic TIL population.

In some embodiments, the TIL population is genetically modified to silence or reduce the expression of one or more cell surface receptors. In exemplary embodiments, the cell surface receptors are CD39 and/or CD69. As used herein, "CD39", "ENTPD1", "ATPDase", "NTPDase-1", "SPG64" and "extracellular nucleoside triphosphate diphosphate hydrolase 1" all refer to the catalytic nucleus triphosphate Cell surface enzyme that hydrolyzes γ- and β-phosphate residues of glycosides and nucleoside diphosphates to nucleoside monophosphate derivatives. High expression or activity of CD39 prevents the immune system from suppressing cancer progression. As used herein, "CD69", "AIM", "BL-AC/P26", "CLEC2C", "EA1", "GP32/28", "MLR-3" all refer to the human Transmembrane C-type lectin protein. CD69 is an activation marker expressed in many cell types in the immune system and is involved in lymphocyte proliferation and signal transmission in lymphocytes. Thus, without being bound by any particular theory of operation, it is believed that TILs genetically modified to silence or reduce the expression of CD39 and CD69 exhibit increased anti-tumor activity. TILs can be modified to silence or reduce CD39 and CD69 expression using any suitable method known in the art, including the genetic modification methods described herein. Exemplary gene modification techniques include, for example, CRISPR, TALE, and zinc finger methods as described herein.

In some embodiments, the genetically modified TIL population is first preselected for CD39/CD69 double negative expression, and then enriched for CD39/CD69 double negative TIL population genetically modified to silence or reduce CD39 and CD69 expression. Without being bound by any particular theory of operation, it is believed that such enriched CD39/CD69 double negative TIL populations, which are then genetically modified to silence or reduce CD39 and CD69 expression, are compared to control TIL populations (e.g., not directed against CD39/CD69 double negative TIL populations expressing preselected and/or subsequently modified to reduce CD39 and CD69 expression exhibit enhanced anti-tumor activity. TILs are preselected for CD39/CD69 double negative expression using any suitable method, including, for example, the CD39/CD69 double negative preselection method provided herein.

In some embodiments, the population of genetically modified TILs with silenced or reduced expression of CD39 and CD69 are then expanded to generate a population of therapeutic TIL populations genetically modified to silence or reduce expression of CD39 and CD69. The population of genetically modified TILs can be amplified using any suitable amplification method.

A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any of the embodiments of the methods described herein, wherein the method further comprises gene editing at least a portion of the TILs. According to other embodiments, the method for expanding TILs into a population of therapeutic TILs is performed according to any of the embodiments of the methods described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, which is described in its entirety Incorporated herein by reference, wherein the method further comprises gene editing at least a portion of the TIL. Accordingly, embodiments of the invention provide a population of therapeutic TILs that has been expanded according to any of the embodiments described herein, wherein at least a portion of the therapeutic population has been gene edited, e.g., at least a portion of the population of therapeutic TILs transferred to the infusion bag has been permanently Sex gene editing. B. Timing of gene editing during TIL expansion

In some embodiments, TIL populations are genetically modified during the expansion methods provided herein. Amplification methods (eg, the 2A and Gen 3 processes described herein or the process depicted in Figure 34) generally include a first amplification and a second amplification. In certain embodiments, TILs are preselected for CD39/CD69 double negative expression prior to the first expansion of the expansion method. In some embodiments, this enrichment for CD39/CD69 is preceded by a first amplification (e.g., a 2A or Gen 3 process first amplification as described herein or the first amplification depicted in FIG. 34 ) The double negative population is genetically modified to silence or minimize CD39 and CD69 expression. In some embodiments, a CD39- and CD69-enriched population undergoes a first amplification and is subjected to a second amplification (e.g., a 2A or Gen 3 process second amplification as described herein or depicted in FIG. 34 ). Prior to the first expansion), the cells generated in the first expansion were genetically modified to silence or reduce CD39 and CD69 expression. In some embodiments, a population enriched for CD39/CD69 double negatives undergoes a first expansion and a second expansion, and the TILs produced by the second expansion are genetically modified to silence or reduce CD39 and CD69 expression.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining from a sample containing a mixture of tumor and TIL cells from a cancer in a patient or individual and / or accept the first TIL population; (b) select CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL from the first TIL population in (a) to obtain enriched CD39/CD69 double negative and/or CD39 TIL population of ^LO /CD69 ^LO ; (c) Enrichment of CD39/CD69 double negative and/or CD39 ^LO /CD69 by culturing in primary cell culture medium containing IL-2, OKT-3 and antigen presenting cells (APCs) The TIL population of ^{the LO} is subjected to a first amplification to produce a second TIL population, wherein the initial first amplification is carried out in a container comprising a first gas-permeable surface area, wherein the initial first amplification is performed for about 1 to 11 A first period of days to obtain a second TIL population, wherein the number of the second TIL population is greater than the first TIL population; (d) by culturing the second TIL in a second culture medium comprising IL-2, OKT-3 and APC population to undergo a rapid second expansion to produce a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain the treatment (e) collecting a third TIL population; and (f) genetically modifying at any time during the method to enrich for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL populations such that the collected third TIL population The population of TILs comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69.

Any population of TILs in the method can be subjected to the genetic modification process as described in step (f) of the examples described above, which means that the TILs can be genetically modified before, during or after any step in the amplification method Editing; for example, during any of steps (a)-(e) outlined in the above method. According to certain embodiments, TILs are collected during the expansion method and the collected TILs undergo a gene editing process and, in some cases, are then reintroduced back into the expansion method (e.g., back into the culture medium) to continue the expansion process, At least a portion of the therapeutic TIL population is rendered permanently gene edited. In some embodiments, the genetic modification process can be performed prior to amplification by activating TILs, performing a gene editing step on the activated TILs, and amplifying the gene-edited TILs according to the processes described herein.

It should be noted that alternative embodiments of the amplification process may differ from the methods shown above; for example, alternative embodiments may not have the same steps (a)-(g), and may have a different number of steps. Regardless of the particular embodiment, the gene editing process can be performed at any time during the TIL expansion method. For example, alternative embodiments may include more than two amplifications, with the possibility of performing a genetic modification step on the TIL during a third or fourth amplification, etc.

According to some embodiments, the genetic modification process is performed on TILs from the CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population, the second population and the third population. For example, CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL populations or a portion of TILs collected from CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO populations can be genetically modified, and during the gene editing process These TILs can then be placed back into the expansion process (eg, back into the culture medium) thereafter. Alternatively, TILs from the second or third population, or a portion of TILs collected from the second or third population, respectively, can be gene edited, and after the gene editing process, these TILs can then be placed back into the amplification process (eg, placed back into culture medium). According to other embodiments, the genetic modification is performed while the TIL is still in the medium and while the expansion is in progress, ie, the gene editing can be performed without "removing" the TIL from the expansion.

According to other embodiments, the genetic modification process is performed on the TILs from the first amplification or the TILs from the second amplification or both. For example, during the first amplification or the second amplification, TILs collected from the culture medium can be genetically modified and after the gene editing process, these TILs can then be placed back into the amplification method, for example by It was reintroduced back into the medium.

According to other embodiments, at least a portion of the TILs are subjected to a genetic modification process after the first amplification and before the second amplification. For example, after the first amplification, TILs collected from the culture medium can be gene edited, and after the genetic modification process, these TILs can then be placed back into the amplification method (e.g., by reintroducing them back into culture medium) for the second amplification.

According to an alternative embodiment, before step (c) (e.g., before, during or after any of steps (a)-(b)), before step (d) (e.g., after step (a) - carrying out the gene editing process before, during or after any of (c)) or before step (e) (eg, before, during or after any of steps (a)-(d)).

In other embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining tumor fragments from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments. The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL were selected from the first TIL population in (a) , to obtain a CD39/CD69 double-negative TIL population enriched; (d) by including IL-2 and optionally OKT-3 (for example, OKT-3 can be present in the culture medium at the beginning of the expansion process) Middle) the first population of TILs is cultured in cell culture medium for a first expansion to produce a second population of TILs, wherein the first expansion is performed in a closed vessel providing a first gas-permeable surface area, wherein the first expansion is performed about 3-14 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is carried out without opening the system; (e) by using additional IL-2, optionally The selected OKT-3 and antigen-presenting cells (APC) are supplemented with the cell culture medium of the second TIL population for second expansion to produce a third TIL population, wherein the second expansion is carried out for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally at (f) collecting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) is optionally performed without opening the system; ( g) transferring the collected TIL population from step (e) to an infusion bag, wherein the transfer from steps (e) to (f) is optionally performed without opening the system; and (h) optionally in Genetic modification at any time prior to transferring the infusion bag in step (g) enriches CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations such that the collected TIL populations comprise genetically modified TILs that were Genetically modified TILs include genetic modifications that reduce the expression of CD39 and CD69.

As described in step (g) of the examples described above, the gene editing process can be performed at any time during the TIL expansion method in step (f) prior to transfer to the infusion bag, which means that it can be performed during the amplification Gene editing of TILs before, during or after any step in the method; for example, during any of steps (a)-(f) outlined in the method above or during a step outlined in the method above Before or after any of (a)-(e). According to certain embodiments, TILs are collected during the amplification process (e.g., the amplification process is "paused" for at least a portion of the TILs) and the collected TILs are subjected to the gene editing process and, in some cases, then reintroduced back into the amplification process methods (eg, introduction back into culture medium) to continue the expansion process such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene edited. In some embodiments, the gene editing process can be performed prior to amplification by activating TILs, performing a gene editing step on the activated TILs, and amplifying the gene edited TILs according to the processes described herein.

It should be noted that alternative embodiments of the amplification process may differ from the methods shown above; for example, alternative embodiments may not have the same steps (a)-(g), and may have a different number of steps. Regardless of the particular embodiment, the gene editing process can be performed at any time during the TIL expansion method. For example, alternative embodiments may include more than two amplifications, with the possibility of gene editing of TILs during a third or fourth amplification, etc.

According to some embodiments, the gene editing process is performed on TILs from one or more of the first population, the second population and the third population. For example, the first population of TILs or a portion of the collected TILs from the first population can be gene edited, and following the gene editing process, these TILs can then be placed back into the amplification process (e.g., placed in returned to the medium). Alternatively, TILs from the second or third population, or a portion of collected TILs from the second or third population, respectively, can be gene edited, and after the gene editing process, these TILs can then be placed back into the expanded during growth (e.g., placement back into culture medium). According to other embodiments, the gene editing is performed while the TILs are still in the medium and while the expansion is in progress, ie, the gene editing can be performed without "removing" the TILs from the expansion.

According to other embodiments, the gene editing process is performed on TILs from the first amplification or TILs from the second amplification or both. For example, during the first amplification or the second amplification, TILs collected from the culture medium can be gene edited and after the gene editing process, these TILs can then be placed back into the amplification method, for example by It was reintroduced back into the medium.

According to other embodiments, at least a portion of the TILs are subjected to a gene editing process after the first amplification and before the second amplification. For example, after the first amplification, TILs collected from the culture medium can be gene edited, and after the gene editing process, these TILs can then be placed back into the amplification method (e.g., by reintroducing them back into culture medium) for the second amplification.

According to an alternative embodiment, before step (c) (e.g., before, during or after any of steps (a)-(b)), before step (d) (e.g., after step (a) - before, during or after any of (c)), before step (e) (for example, before, during or after steps (a)-(d)) or before step (f) (for example, Before, during or after any of steps (a)-(e)) the gene editing process is performed.

It should be noted that with respect to OKT-3, according to certain embodiments, the cell culture medium may start to include OKT-3 at the beginning of the first expansion (day 0) or day 1, such that at day 0 and/or day 1 1 day, TILs were gene-edited after they had been exposed to OKT-3 in cell culture medium. According to other embodiments, the cell culture medium comprises OKT-3 during the first expansion and/or during the second expansion, and the gene editing is performed prior to the introduction of OKT-3 into the cell culture medium. Alternatively, the cell culture medium can comprise OKT-3 during the first expansion and/or during the second expansion, and the gene editing is performed after introducing OKT-3 into the cell culture medium.

It should also be noted that, with respect to 4-1BB agonists, according to certain embodiments, the cell culture medium may include the 4-1BB agonist starting on the day of initiation of the first expansion (day 0) or day 1 such that at On day 0 and/or day 1, TILs are gene edited after they have been exposed to the 4-1BB agonist in the cell culture medium. According to other embodiments, the cell culture medium comprises a 4-1BB agonist during the first expansion and/or during the second expansion, and the gene editing is performed prior to introducing the 4-1BB agonist into the cell culture medium. Alternatively, the cell culture medium can comprise 4-1BB during the first expansion and/or during the second expansion, and the gene editing is performed after introducing 4-1BB into the cell culture medium.

It should also be noted that with respect to IL-2, according to certain embodiments, the cell culture medium may start to include IL-2 at the beginning of the first expansion (day 0) or day 1, such that at day 0 and/or day 1 One day, TILs were gene edited after they had been exposed to IL-2 in cell culture medium. According to other embodiments, the cell culture medium comprises IL-2 during the first expansion and/or during the second expansion, and gene editing is performed prior to introducing IL-2 into the cell culture medium. Alternatively, the cell culture medium can comprise IL-2 during the first expansion and/or during the second expansion, and the gene editing is performed after the introduction of IL-2 into the cell culture medium.

As discussed above, the cell culture medium can include one or more of OKT-3, a 4-1BB agonist, and IL-2 beginning on day 0 or day 1 of the first expansion. According to some embodiments, the cell culture medium includes OKT-3 starting on day 0 or day 1 of the first expansion, and/or the cell culture medium includes 4-1BB promoter starting on day 0 or day 1 of the first expansion. Efficacy agent, and/or cell culture medium includes IL-2 beginning on day 0 or day 1 of the first expansion. According to one example, the cell culture medium comprises OKT-3 and the 4-1BB agonist starting at day 0 or day 1 of the first expansion. According to another example, the cell culture medium comprises OKT-3, the 4-1BB agonist and IL-2 starting at day 0 or day 1 of the first expansion. Of course, one or more of OKT-3, 4-1BB agonists, and IL-2 can be added to the cell culture medium at one or more other time points during the expansion process, as described in various embodiments herein. explained in the example.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL were selected from the first TIL population in (a), To obtain a CD39/CD69 double-negative TIL population enriched; (d) by culturing the first TIL population in a cell culture medium comprising IL-2 and, optionally, OKT-3 and/or 4-1BB agonist antibodies for about 3 to 11 days to carry out the first expansion to produce the second TIL population, wherein the first expansion is carried out in a closed container providing the first air-permeable surface area; (e) by adding OKT-3 and culturing for about 1 Stimulating the second TIL population for at least 3 days, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) sterile electroporating the second TIL population to achieve transferring at least one gene editor into a portion of the cells of the second TIL population; (g) resting the second TIL population for about 1 day; (h) by using additional IL-2, optionally OKT-3 Antibody, optionally OX40 antibody, and antigen presenting cells (APCs) supplement the cell culture medium of the second TIL population for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain A third TIL population, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (g) to step (h) is performed without opening the system; (i ) collecting the therapeutic TIL population obtained from step (h) to obtain a collected TIL population, wherein the transition from step (h) to step (i) is performed without opening the system, wherein the collected TIL population is a therapeutic TIL population; and (j) transferring the collected TIL population to an infusion bag, wherein the transfer from steps (h) to (i) is optionally performed without opening the system, wherein at least one gene Sterile electroporation of the editor into the fraction of the cells of the second TIL population can modulate the plurality of cells in the fraction to reduce the expression of CD39 and CD69.

According to some embodiments, the foregoing method further comprises cryopreserving the collected TIL population using a cryopreservation medium. In some embodiments, the cryopreservation medium is a dimethylsulfone-based cryopreservation medium. In other embodiments, the cryopreservation medium is CS10.

In other embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining and/or receiving a first TIL population from a sample containing a mixture of tumor and TIL cells from a cancer in a patient or individual; (b) selecting CD39/CD69 double negative TILs from the first TIL population in (a), to obtain a CD39/CD69 double negative TIL population enriched; (c) Initiating a first expansion by culturing a TIL population enriched for CD39/CD69 double negatives in a first cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to generate a second Two TIL populations, wherein the initial first expansion is performed in a container comprising a first gas-permeable surface area, wherein the initial first expansion is performed for a first period of about 1 to 11 days to obtain a second TIL population, wherein the first The number of the second TIL population is greater than the first TIL population; (d) Optionally restimulate a second TIL population with OKT-3; (e) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified TIL population comprises a genetic modification that reduces the expression of CD39 and CD69; (f) rapid second expansion by culturing the modified second TIL population in a second medium comprising IL-2, OKT-3, and APCs to produce a third TIL population, wherein the rapid second expansion performing a second period of about 14 days or less to obtain a therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population comprising a genetic modification that reduces the expression of CD39 and CD69; and (g) Collect the third TIL population.

In some embodiments, the genetic modification step comprises electroporation and delivery of at least one gene editor system selected from the group consisting of: clustered regularly interspaced short palindromic repeat (CRISPR) system, transcription activator-like effector (TALE ) system or zinc finger system, wherein the at least one gene editor system reduces the expression of CD39 and/or CD69 in the modified second TIL population.

According to some embodiments, the foregoing methods may be used to provide an autologously collected population of TILs for use in the treatment of human subjects with cancer. C. Gene Editing Methods

As discussed above, embodiments of the present invention provide tumor infiltrating lymphocytes (TILs) that have been genetically modified through gene editing such that they enhance therapeutic effects (eg, expression of immunomodulatory fusion proteins on their cell surface). Embodiments of the invention encompass gene editing via nucleotide insertion (RNA or DNA) into TIL populations to promote expression of one or more proteins and repress expression of one or more proteins and combinations thereof Embodiments of the invention also provide Methods for expanding TILs into a therapeutic population, wherein the methods comprise gene editing TILs. There are several gene editing techniques available for genetically modifying TIL populations that are suitable for use in accordance with the present invention.

In some embodiments, methods of genetically modifying a population of TILs include the step of stably incorporating genes for the production of one or more proteins. In one embodiment, the method of genetically modifying a population of TILs includes the step of retroviral transduction. In some embodiments, the method of genetically modifying a population of TILs includes the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in: Levine et al., Proceedings of the National Academy of Sciences USA 2006, 103, 17372-77; Zufferey et al., Nature Biotechnology 1997 , 15, 871-75; Dull et al., Journal of Virology 1998, 72, 8463-71 and US Patent No. 6,627,442, the respective disclosures of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes the step of gamma-retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, "Cur. Prot. Mol. Biol." 1996, 9.9.1-9.9 .16, the disclosure of which is incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include those in which the translocase is provided as a DNA expression vector or as expressible RNA or protein so that long-term expression of the translocase does not occur in the transgene In cells, for example, a translocase is provided as mRNA (eg, mRNA comprising a cap and a polyA tail). Suitable transposon-mediated gene transfer systems including salmon-like Tel-like translocases (SB or Sleeping Beauty translocases), such as SB10, SB11, and SB100x; and engineered enzymes with increased enzymatic activity are described, for example, below Middle: Hackett et al., Mol. Therapy 2010, 18, 674-83 and US Patent No. 6,489,458, the respective disclosures of which are incorporated herein by reference.

In some embodiments, methods of genetically modifying a population of TILs include the step of stably incorporating genes for the production or repression (eg, silencing) of one or more proteins. In some embodiments, the method of genetically modifying a population of TILs includes the step of electroporation. Electroporation methods are known in the art and are described, for example, in Tsong, Biophysical Journal 1991, 60 , 297-306 and US Patent Application Publication No. 2014/0227237 A1, their respective disclosures The contents are incorporated herein by reference. Other electroporation methods known in the art, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, Nos. 5,304,120, 5,318,514, 6,010,613 and 6,078,490, the disclosures of which are incorporated herein by reference. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising applying a A series of at least three single, operator-controlled steps of independently programmed DC electrical pulses with field strengths equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two or three of the following characteristics (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different in pulse width from each other; and (3) the first group of the at least A first pulse interval of two of the three pulses is different from a second pulse interval of two of the second set of at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising applying a The step of a series of at least three single, operator-controlled independently programmed DC electrical pulses having a field strength equal to or greater than 100 V/cm, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising applying a The step of a series of at least three single, operator-controlled independently programmed DC electrical pulses having a field strength equal to or greater than 100 V/cm, wherein at least two of the at least three pulses differ from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising applying a A series of steps of at least three single, operator-controlled independently programmed DC electrical pulses having a field strength equal to or greater than 100 V/cm, wherein the first pulse interval of two of the at least three pulses in the first set is separated from the second The second pulse intervals of two of the set of at least three pulses are different. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to induce pore formation in the TIL, comprising the step of applying a series of at least three DC electrical pulses to the TIL at a field strength equal to or Greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) At least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two of the at least three pulses in the first group and the interval between the two of the at least three pulses in the second group The second pulse interval was varied such that the induced pores lasted for a relatively long period of time and so that the viability of the TIL was maintained.

In some embodiments, the method of genetically modifying a population of TILs comprises the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating and endocytosis) are known in the art and described in: Graham and van der Eb, Virology 1973 , 52 , 456- 467; Wigler et al., Proceedings of the National Academy of Sciences USA 1979 , 76 , 1373-1376; and Chen and Okayarea, Molecular Cell Biology 1987 , 7 , 2745-2752; and US Patent No. 5,593,875, each of which The disclosure is incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs comprises the step of lipofection. Lipofectamine methods, such as the use of cationic lipids N- [1-(2,3-dioleyloxy)propyl] -n , n , n -trimethylammonium chloride (DOTMA) and dioleyl Methods for 1:1 (w/w) liposome formulations of phosphatidylethanolamine (DOPE) in filtered water are known in the art and described in: Rose et al., Biotechnology 1991 , 10 , 520-525 and Felgner et al., Proceedings of the National Academy of Sciences, 1987 , 84 , 7413-7417 and US Patent Nos. 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484 and 7,687,070 , the respective disclosures of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes the step of transfection using the methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, each of The disclosure is incorporated herein by reference.

According to one embodiment, the gene editing method may comprise the use of programmable nucleases that mediate double-stranded or single-stranded breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific gene body loci, i.e., they rely on recognizing a specific DNA sequence within the gene body to target the nuclease domain to this location and mediate Generates a double-stranded break at the target sequence. Double-stranded breaks in DNA then recruit endogenous repair mechanisms to the site of the break to mediate genome editing by non-homologous end joining (NHEJ) or homology-directed repair (HDR). Thus, repair of the break can result in the introduction of insertion/deletion mutations that disrupt (eg, silence, suppress, or enhance) the gene product of interest.

Major classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-related nucleases. Nucleases (eg CRISPR/Cas9). These nuclease systems can be roughly classified into two categories based on their DNA recognition modes: ZFNs and TALENs achieve specific DNA binding through protein-DNA interactions, while CRISPR systems, such as Cas9, RNA guides molecules and targets specific DNA sequences through protein-DNA interactions. See eg Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene editing methods that can be used in accordance with the TIL expansion methods of the present invention include CRISPR methods, TALE methods, and ZFN methods, examples of which are described in more detail below. According to some embodiments, the method of expanding TILs into a therapeutic population can be according to any embodiment of the methods described herein (e.g. Method 2A) or as described in PCT/US2017/058610, PCT/US2018/012605 or PCT/US2018/012633 wherein the method further comprises gene editing at least a portion of the TILs by one or more of a CRISPR approach, a TALE approach, or a ZFN approach to generate TILs that provide enhanced therapeutic effects. According to some embodiments, gene editing can be assessed by comparing gene-edited TILs with unmodified TILs in vitro, e.g., by assessing in vitro effector functions, cytokine profiles, etc. compared to unmodified TILs Improved therapeutic effect of TIL.

In some embodiments of the invention, electroporation is used to deliver gene editing systems, such as CRISPR, TALEN and ZFN systems. In some embodiments of the invention, the electroporation system is a flow electroporation system. An example of a suitable flow electroporation system suitable for some embodiments of the invention is the commercially available MaxCyte STX system. There are several alternative commercially available electroporation instruments that may be suitable for use in the present invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Celelectricon), Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax) or siPORTer96 (Ambion). In some embodiments of the invention, the electroporation system forms a closed sterile system together with the rest of the TIL amplification method. In some embodiments of the invention In embodiments, the electroporation system is a pulsed electroporation system as described herein, and together with the remainder of the TIL expansion method forms a closed sterile system. a. CRISPR method

The method for expanding TILs into a therapeutic population can be according to any of the examples of the methods described herein (e.g. Process 2A) or as described in PCT/US2017/058610, PCT/US2018/012605 or PCT/US2018/012633 is described, wherein the method further comprises gene editing at least a portion of the TIL by a CRISPR method (eg, CRISPR/Cas9 or CRISPR/Cpf1). According to certain embodiments, the use of CRISPR methods during the TIL expansion process can result in the expression of at least one immune modulator at the cell surface of at least a portion of the therapeutic TIL population and optionally the silencing or reduction of one or more immune checkpoint genes. Alternatively, the use of CRISPR methods during the TIL expansion process can result in the expression of at least one immunomodulatory composition at the cell surface of at least a portion of the therapeutic TIL population and optionally the enhancement of one or more immune checkpoint genes. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, and IL-21.

CRISPR stands for "Clustered Regularly Interspaced Short Palindromic Repeats". Methods of gene editing using the CRISPR system are also referred to herein as CRISPR methods. CRISPR systems can be divided into two main categories, Type 1 and Type 2, which are further divided into different types and subtypes. The classification of CRISPR systems is based on effector Cas proteins capable of cleaving specific nucleic acids. In type 1 CRISPR systems, the effector module consists of a multiprotein complex, whereas type 2 systems use only one effector protein. Type 1 CRISPRs include types I, III, and IV, and type 2 CRISPRs include types II, V, and VI. Although any of these types of CRISPR systems can be used in accordance with the present invention, there are three types of CRISPR systems that incorporate RNA and Cas proteins that are preferred for use in accordance with the present invention: I (exemplified by Cas3), II (exemplified by Cas9) and type III (exemplified by Cas10). Type II CRISPR is one of the best characterized systems.

CRISPR technology is adapted from the natural defense mechanisms of bacteria and archaea (the domain of single-celled microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins, including Cas9, to block the attack of viruses and other exosomes by shredding and destroying the DNA of foreign invaders. CRISPR is a specialized region of DNA with two unique features: the presence of nucleotide repeats and spacers. Repeats of nucleotides are distributed throughout the CRISPR region, with short foreign DNA segments (spacers) interspersed in the repeats. In type II CRISPR/Cas systems, spacers are integrated within the CRISPR gene body locus and are transcribed and processed into short CRISPR RNA (crRNA). These crRNAs anneal to transactivating crRNAs (tracrRNAs) and direct Cas proteins for sequence-specific cleavage and silencing of pathogenic DNA. Target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA binding region. Thus the CRISPR/Cas system can be retargeted to cleave almost any DNA sequence by redesigning crRNA. Thus, according to certain embodiments, Cas9 acts as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA recognition. The crRNA and tracrRNA in the native system can be simplified to a single guide RNA (sgRNA) of about 100 nucleotides for genetic engineering. sgRNAs are synthetic RNAs that include the backbone sequence necessary for Cas binding and a user-defined approximately 17- to 20-nucleotide spacer that defines the gene body target to be modified. Thus, users can alter the genome target of Cas proteins by altering the target sequence present in the sgRNA. Human cells can directly host the CRISPR/Cas system by co-delivering plastids expressing the Cas9 endonuclease and RNA components (eg, sgRNA). Different Cas protein variants can be used to reduce targeting limitations (eg heterologs of Cas9 such as Cpf1).

According to some embodiments, an engineered, programmable, non-naturally occurring Type II CRISPR-Cas system comprises a Cas9 protein and at least one guide RNA that targets and hybridizes to a target sequence of a DNA molecule in a TIL , wherein the DNA molecule encodes and the TIL expresses at least one immune checkpoint molecule, and the Cas9 protein cleaves the DNA molecule, thereby altering the expression of the at least one immune checkpoint molecule; and wherein the Cas9 protein and the guide RNA do not naturally co-exist . According to one embodiment, the expression of two or more immune checkpoint molecules is altered. According to one embodiment, the guide RNA comprises a guide sequence fused to a tracr sequence. For example, guide RNA can comprise crRNA-tracrRNA or sgRNA. According to aspects of the invention, the terms "guide RNA", "single guide RNA" and "synthetic guide RNA" are used interchangeably and refer to a polynucleotide sequence comprising a guide sequence within a guide RNA specifying a target site. About 17-20 bp sequence.

According to embodiments of the present invention, variants of Cas9 with improved on-target specificity compared to Cas9 may also be used. Such variants may be referred to as high-fidelity Cas-9. According to one embodiment, a double nickase approach can be utilized, wherein two nickases targeting opposing DNA strands generate a DSB within the target DNA (commonly referred to as double nick or double nickase CRISPR systems). For example, this approach can involve mutations in one of the two Cas9 nuclease domains, converting Cas9 from a nuclease to a nickase. Non-limiting examples of high-fidelity Cas9 include eSpCas9, SpCas9-HF1, and HypaCas9. Such variants can reduce or eliminate undesired changes at non-target DNA sites. See, eg, Slaymaker IM et al., Science . 1 Dec. 2015 , Kleinstiver BP et al., Nature . 6 Jan. 2016 , and Ran et al., Handbook of Natural Experiments ( Nat Protoc. ) " 2013 Nov; 8(11): 2281-2308, the disclosure of which is incorporated herein by reference.

Furthermore, according to certain embodiments, improved gene delivery of Cas9 into cells and improved on-target specificity can be used with Cas9 backbones such as those disclosed in U.S. Patent Application Publication No. 2016/0102324, which is incorporated by reference In this article. For example, the Cas9 backbone can include a RuvC motif as defined by (D-[I/L]-G-X-X-S-X-G-W-A) and/or a HNH motif as defined by (Y-X-X-D-H-X-X-P-X-S-X-X-X-D-X-S), where X represents 20 naturally occurring amino acids Any of them and [I/L] means isoleucine or leucine. The HNH domain is responsible for cleaving one strand of the target dsDNA and the RuvC domain is involved in the cleavage of the other strand of dsDNA. Thus, each of these domains cleaves one strand of the target DNA within the spacer immediately preceding the PAM, causing blunt cleavage of the DNA. These motifs can be combined with each other to generate a more compact and/or more specific Cas9 backbone. In addition, the motif can be used to generate a split Cas9 protein that splits into two separate RuvC and HNH domains (i.e., a reduced or truncated version of the Cas9 protein or Cas9 variant that contains either the RuvC domain or the HNH domain), which Target DNA can be processed together or separately.

According to certain embodiments, the CRISPR method comprises silencing or reducing the expression of one or more immune checkpoint genes in TILs by introducing a Cas9 nuclease and a guide RNA (e.g., crRNA-tracrRNA or sgRNA) containing a gene for the immune checkpoint. The target DNA sequence of the point gene has a specific sequence of about 17-20 nucleotides. The guide RNA can be delivered in the form of RNA or by transforming a plastid with the guide RNA coding sequence according to the promoter. Based on the target sequence defined by the sgRNA, the CRISPR/Cas enzyme introduces a double-stranded break (DSB) at a specific position. DSBs in cells can be repaired by non-homologous end joining (NHEJ, a mechanism that normally causes insertions or deletions (indels) in DNA). Insertions/deletions often cause a reading frame shift, resulting in a loss-of-function allele; for example, by introducing a premature stop codon within the open reading frame (ORF) of the gene of interest. According to certain embodiments, the result is a loss-of-function mutation in an immune checkpoint gene of interest.

Alternatively, instead of NHEJ, DSBs induced by CRISPR/Cas enzymes can be repaired by homology-directed repair (HDR). While NHEJ-mediated DSB repair typically disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes ranging from single nucleotide changes to large insertions. According to one embodiment, HDR is used to gene edit immune checkpoint genes by delivering DNA repair templates containing desired sequences into TILs with sgRNA and Cas9 or Cas9 nickase. The repair template preferably contains the desired edit and other homologous sequences (often referred to as left and right homology arms) immediately upstream and downstream of the gene of interest.

According to certain embodiments, an enzymatically inactive form of Cas9 (deadCas9 or dCas9) can be targeted to the transcription initiation site to inhibit transcription by blocking initiation. Thus, immune checkpoint genes of interest can be inhibited without the use of DSBs. The dCas9 molecule retains the ability to bind to target DNA based on the sgRNA targeting sequence. According to an embodiment of the present invention, the CRISPR method comprises silencing or reducing the expression of one or more immune checkpoint genes by inhibiting or preventing the transcription of target genes. For example, a CRISPR approach may involve fusing a transcriptional repressor domain, such as a Kruppel-associated box (KRAB) domain, to an enzymatically inactive form of Cas9, thereby forming, for example, dCas9-KRAB, which targets the transcription of immune checkpoint genes Initiation site, which causes repression or prevents the transcription of a gene. Preferably, the repressor domain is targeted to a window downstream of the transcription initiation site, eg, about 500 bp downstream. This approach, which may be referred to as CRISPR interference (CRISPRi), results in stable gene attenuation through reduced transcription of the target RNA.

According to certain embodiments, an enzymatically inactive form of Cas9 (deadCas9 or dCas9) can be targeted to the transcription initiation site to activate transcription. This method may be referred to as CRISPR activation (CRISPRa). According to one embodiment, the CRISPR method comprises increasing the expression of one or more immune checkpoint genes by activating transcription of the target gene. According to such embodiments, immune checkpoint genes of interest can be activated without the use of DSBs. The CRISPR approach can involve targeting a transcriptional activation domain to a transcriptional start site; for example, by fusing a transcriptional activator such as VP64 to dCas9, thereby forming, for example, dCas9-VP64, which targets immune checkpoint genes Transcription initiation site, which causes the activation of gene translation. Preferably, the activator domain is targeted to a window upstream of the transcription start site, eg, about 50-400 bp downstream.

Other embodiments of the invention may utilize activation strategies developed for potent activation of target genes in mammalian cells. Non-limiting examples include co-expression of epitope-tagged dCas9 and antibody-activator effector proteins (e.g., the SunTag system), dCas9 fused to multiple distinct tandem activation domains (e.g., dCas9-VPR), or with Co-expression of dCas9-VP64 with modified backbone gRNAs and other RNA-binding coactivators (eg, SAM activators).

According to other embodiments, a CRISPR-mediated genome editing method known as CRISPR-assisted rational protein engineering (CARPE), as disclosed in U.S. Patent No. 9,982,278, which is incorporated by reference, may be used in accordance with embodiments of the present invention incorporated into this article. CARPE involves the generation of "donor" and "destination" libraries that incorporate directed mutations from single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) editing cassettes directly into the genome. The construction of the donor library involves the co-transformation of rationally designed editing oligonucleotides and guide RNA (gRNA) that hybridizes to the target DNA sequence into cells. Editing oligonucleotides are designed to couple a deletion or mutation of a PAM with a mutation of one or more desired codons in an adjacent gene. This enables the generation of an entire donor pool in a single transformation. The donor pool is searched by amplification of the recombinant chromosome, such as by a PCR reaction, using synthetic features from the editing oligonucleotide, i.e., a second PAM deletion or mutation incorporated simultaneously at the 3' end of the gene . This enables covalent coupling of codon-targeted mutations for PAM deletions. Next, the donor repertoire is co-transformed into cells with the gRNA vector of interest to generate a population of cells expressing the rationally designed protein repertoire.

According to other embodiments, a system for using CRISPR-mediated methods called Genome Engineering by Traceable CRISPR-Enriched Recombination Engineering (GEn-TraCER) may be used in accordance with embodiments of the present invention for traceable, Methods of precise genome editing are disclosed in US Patent No. 9,982,278, which is incorporated herein by reference. The GEn-TraCER method and vectors combine the editing cassette and the gene encoding the gRNA on a single vector. Cassettes contain desired mutations as well as PAM mutations. A vector that also encodes Cas9 is introduced into the cell or population of cells. Expression of this activated CRISPR system in the cell or cell population causes the gRNA to recruit Cas9 to the target region where dsDNA breaks occur, enabling the integration of the PAM mutation.

Non-limiting examples of genes that can be silenced or suppressed by permanent gene editing of TILs via the CRISPR approach include CD39, CD69, PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ , PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7 , FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3 and TOX.

Non-limiting examples of genes that can be enhanced by permanent gene editing of TILs via CRISPR methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL -15, IL-18, IL-21, NOTCH 1/2 intracellular domain (ICD), and/or NOTCH ligand mDLL1.

Examples of systems, methods, and compositions that utilize the CRISPR approach to alter the expression of a gene sequence of interest and that can be used in accordance with embodiments of the present invention are described in U.S. Patent Nos. 8,697,359, 8,993,233, 8,795,965, 8,771,945, 8,889,356, 8,865,406, 8,999,641, 8,945,839, 8,932,814, 8,871,445, 8,906,616, and 8,895,308, which are incorporated herein by reference. Resources for performing CRISPR methods, such as plasmids for expressing CRISPR/Cas9 and CRISPR/Cpf1, are commercially available from companies such as GenScript.

In some embodiments, genetic modification of TIL populations as described herein can be performed using the CRISPR/Cpf1 system as described in US Pat. No. 9,790,490, the disclosure of which is incorporated herein by reference. The CRISPR/Cpf1 system differs functionally from the CRISPR-Cas9 system in that no additional tracrRNA is required to process the Cpf1-associated CRISPR array into mature crRNA. The crRNA used in the CRISPR/Cpf1 system has a spacer or guide sequence and a direct repeat sequence. The Cpf1p-crRNA complex formed by this method alone is sufficient to cleave the target DNA.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system as appropriate; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative were selected from the first TIL population in (a) TILs to obtain a population of TILs enriched for CD39/CD69 double negatives; (d) by culturing the first TILs in cell culture medium comprising IL-2 and optionally OKT-3 and/or 4-1BB agonist antibodies The population is approximately 3 to 11 days old for a first expansion to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first air-permeable surface area; (e) by adding OKT-3 and culturing stimulating the second TIL population for about 1 to 3 days, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) sterile electroporating the second TIL population, to achieve the transfer of at least one gene editor to a plurality of cells in the second TIL population; (g) resting the second TIL population for about 1 day; (h) by using additional IL-2, optionally The OKT-3 antibody, the optional OX40 antibody, and antigen-presenting cells (APCs) supplement the cell culture medium of the second TIL population for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, wherein the second expansion is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (g) to step (h) is without opening the system carrying out; (i) collecting the therapeutic TIL population obtained from step (h) to obtain a collected TIL population, wherein the transition from step (h) to step (i) is carried out without opening the system, wherein the The collected TIL population is a therapeutic TIL population; (j) transferring the collected TIL population to an infusion bag, wherein the transfer from steps (i) to (j) is optionally performed without opening the system; and ( k) Cryopreserving the collected TIL population optionally using a cryopreservation medium, wherein the electroporation step comprises delivery of at least one gene editor system selected from the group consisting of: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ Cas9 system and CRISPR/Cpf1 system, wherein the at least one gene editor reduces the expression of CD39 and CD69.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system as appropriate; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative were selected from the first TIL population in (a) TILs to obtain a population of TILs enriched for CD39/CD69 double negatives; (d) by culturing the first TILs in cell culture medium comprising IL-2 and optionally OKT-3 and/or 4-1BB agonist antibodies The population is approximately 3 to 11 days old for a first expansion to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first air-permeable surface area; (e) by adding OKT-3 and culturing Stimulating the second TIL population for about 1 to 3 days to obtain a second TIL population, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) for the second TIL population Sterile electroporation of the TIL population to effect transfer of at least one gene editor into a plurality of cells in a second TIL population; (g) resting the second TIL population for about 1 day; IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) supplement the cell culture medium of the second TIL population for a second expansion to produce a third TIL population, wherein the second TIL population The second expansion is performed for about 7 to 11 days to obtain a third TIL population, wherein the second expansion is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (g) to step (h) is performed without an open system; (i) collecting the therapeutic TIL population obtained from step (h) to obtain a collected TIL population, wherein the transition from step (h) to step (i) is performed in a closed system (j) transferring the collected TIL population to an infusion bag, wherein the transfer from steps (i) to (j) is optionally performed in a non-open system and (k) optionally using a cryopreservation medium to cryopreserve the collected TIL population, wherein the electroporation step comprises delivery of at least one gene editor system selected from the group consisting of clustered regularly spaced short bouts CRISPR/Cas9 system and CRISPR/Cpf1 system, wherein the at least one gene editor system reduces the expression of CD39 and CD69. b. TALE method

The method for expanding TILs into a therapeutic population can be according to any of the examples of the methods described herein (e.g., Process 2A) or as described in PCT/US2017/058610, PCT/US2018/012605 or PCT/US2018/012633 Performed as described, wherein the method further comprises gene editing at least a portion of the TIL by the TALE method. According to certain embodiments, the use of TALE methods during the TIL expansion process can result in the expression of at least one immunomodulatory composition at the cell surface, and optionally one or more immune checkpoint genes in at least a portion of the therapeutic TIL population silence or decrease. Alternatively, use of the TALE approach during the TIL expansion process can result in the expression of at least one immunomodulatory composition at the cell surface and, optionally, enhanced expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

TALE stands for "transcription activator-like effector" proteins, which include TALENs ("transcription activator-like effector nucleases"). Methods of gene editing using the TALE system are also referred to herein as TALE methods. TALEs are naturally occurring proteins from the plant pathogenic bacterium Xanthomonas and contain a DNA-binding domain composed of a series of repeat domains of 33-35 amino acids each recognizing a single base pair. TALE specificity is determined by two hypervariable amino acids called repeat-variable di-residues (RVDs). Modular TALE repeats are linked together to recognize contiguous DNA sequences. Specific RVDs in DNA-binding domains recognize bases in the locus of interest, providing structural features to assemble predictable DNA-binding domains. The DNA binding domain of the TALE was fused to the catalytic domain of the type IIS FokI endonuclease to generate a targetable TALE nuclease. To induce site-specific mutations, two individual TALEN arms separated by a 14-20 base pair spacer region bring Fokl monomers closer together to dimerize and create targeted double-stranded breaks.

Several large systematic studies using various assembly methods indicate that TALE repeats can be incorporated to recognize almost any user-defined sequence. Strategies that enable rapid assembly of custom TALE arrays include Golden Gate molecular selection, high-throughput solid-phase assembly, and ligation-independent selection techniques. Custom-designed TALE arrays were also provided by Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). )) commercially available. In addition, web-based tools such as TAL Effector-Nucleotide Target 2.0 are available, which can design custom TAL effector repeat arrays for desired targets and provide predicted TAL effector binding sites. See Doyle et al., Nucleic Acids Research, 2012 , Vol. 40, W117-W122. TALE and TALEN methods suitable for use in the present invention are described in examples in US Patent Application Publication Nos. US 2011/0201118 A1, US 2013/0117869 A1, US 2013/0315884 A1, US 2015/0203871 No. A1 and No. US 2016/0120906 A1, the disclosures of which are incorporated herein by reference.

According to some embodiments of the present invention, the TALE method comprises silencing or reducing the expression of one or more immune checkpoint genes by inhibiting or preventing the transcription of target genes. For example, a TALE approach may involve the use of KRAB-TALE, wherein the approach involves fusing a transcribed Kruppel-associated box (KRAB) domain to a DNA-binding domain targeting the transcriptional start site of a gene, resulting in repression or prevention of transcription of the gene.

According to other embodiments, the TALE method comprises silencing or reducing the expression of one or more immune checkpoint genes by introducing mutations in the target gene. For example, a TALE approach may involve fusing a nuclease effector domain, such as Fokl, to a TALE DNA binding domain, resulting in a TALEN. Fokl is active as a dimer; thus, the method involves constructing TALEN pairs to localize the FOKL nuclease domains to adjacent gene body target sites where the domains introduce DNA double-strand breaks. Double-strand breakage can be accomplished following correct positioning and dimerization of Fokl. Following the introduction of a double-stranded break, DNA repair can be achieved via two different mechanisms: high-fidelity homologous recombination pairs (HRR) (also known as homology-directed repair or HDR) or error-prone non-homologous end joining (NHEJ). Repair of double-stranded breaks by NHEJ preferably results in deletions, insertions or substitutions of the DNA target site, ie, NHEJ usually results in the introduction of small insertions and deletions at the site of the break, usually inducing a reading frame shift that knocks out gene function. According to certain embodiments, TALEN pairs are targeted to most of the 5' exon of the gene, promoting early frame shift mutations or premature stop codons. Gene mutations introduced by TALENs are preferably permanent. Thus, according to some embodiments, the method comprises silencing or reducing the expression of an immune checkpoint gene by inducing a site-specific double-strand break using a dimerization TALEN, causing one or more mutations in the immune checkpoint gene of interest , the site-specific double-strand break was repaired by error-prone NHEJ.

According to other embodiments, TALENs are used to introduce genetic changes via HRR, such as non-random point mutations, targeted deletions, or additions of DNA segments. Introduction of DNA double-stranded breaks enables gene editing via homologous recombination in the presence of suitable donor DNA. According to some embodiments, the method comprises co-delivering a dimerized TALEN and a donor plastid carrying a locus-specific homology arm to induce a site-specific double-stranded break and integrate one or more transgenes into the DNA.

According to other embodiments, TALENs, which are hybrid proteins derived from FokI and AvrXa7, as disclosed in US Patent Publication No. 2011/0201118, can be used in accordance with embodiments of the present invention. This TALEN retains the recognition specificity for the target nucleotide of AvrXa7 and the double-stranded DNA cleavage activity of FokI. Other TALENs with different recognition specificities can be prepared using the same method. For example, compact TALENs can be generated by engineering core TALE backbones with different sets of RVDs to alter DNA binding specificity and targeting specificity to a single dsDNA target sequence. See US Patent Publication No. 2013/0117869. Catalytic domains of choice can be attached to a backbone to enable DNA processing, which can be engineered to ensure that when fused to the core TALE backbone, the catalytic domain is capable of processing DNA in the vicinity of a single dsDNA target sequence. Peptide linkers can also be engineered to fuse the catalytic domain to the backbone, resulting in a compact TALEN made of a single polypeptide chain that does not require dimerization of a single dsDNA sequence for targeting specificity. The core TALE backbone can also be generated by fusing a catalytic domain (which can be a TAL monomer) to its N-terminus, enabling the possibility that this catalytic domain can interact with another catalytic domain fused to another TAL monomer, thereby generating Modified by catalytic entities that may process DNA near the target sequence. See U.S. Patent Publication No. 2015/0203871. This architecture only allows targeting of one DNA strand, which is not an option for classical TALEN architectures.

According to embodiments of the present invention, conventional RVDs can be used to generate TALENs that can significantly reduce gene expression. In one embodiment, four RVDs, NI, HD, NN, and NG, are used to target adenine, cytosine, guanine, and thymine, respectively. These conventional RVDs can be used, for example, to generate TALENs targeting the PD-1 gene. Examples of TALENs using conventional RVDs include the T3v1 and T1 TALENs disclosed in Gautron et al., Molecular Therapy: Nucleic Acids, Dec. 2017, Vol. 9: 312-321 (Gautron) , which is incorporated herein by reference. T3v1 and T1 TALENs target the second exon of the PDCD1 locus where the PD-L1 binding site is located and can significantly reduce PD-1 production. In one embodiment, T1 TALENs achieve this function by using the target SEQ ID NO:238 and T3v1 TALENs achieve this function by using the target SEQ ID NO:239.

According to other embodiments, TALENs are modified using non-conventional RVDs to improve their activity and specificity against a gene of interest, such as disclosed in Gautron. Naturally occurring RVDs cover only a small fraction of the potentially diverse repertoire of hypervariable amino acid positions. Non-conventional RVDs provide an alternative to native RVDs and have novel inherent target-specific features that can be used to exclude targets outside the TALEN targeting site (sequences within a gene that contain few mismatches relative to the target sequence). Non-conventional RVDs can be identified by generating and screening collections of TALENs containing alternative amino acid combinations at two hypervariable amino acid positions at a given position on the array, as in Juillerat et al., Scientific Reports ( Scientific Reports )" 5, No. 8150 (2015), which is incorporated herein by reference. Next, non-conventional RVDs that can discriminate between nucleotides present at mismatched positions can be selected that prevent TALEN activity at out-of-site sequences while still allowing proper manipulation of the target position. The conventional RVD in the TALEN can then be replaced with the selected non-conventional RVD. Examples of TALENs in which conventional RVDs have been replaced by non-conventional RVDs include T3v2 and T3v3 PD-1 TALENs produced by Gautron. These TALENs have increased specificity compared to TALENs using conventional RVDs.

According to other embodiments, TALENs can be used to introduce genetic changes to silence or reduce the expression of two genes. For example, two independent TALENs can be generated to target two different genes and then used together. The molecular events produced by the two TALENs at their respective loci and potential off-target sites can be characterized by high-throughput DNA sequencing. This enables the analysis of off-target sites and the identification of sites that may result from the use of two TALENs. Based on this information, appropriate conventional and non-conventional RVDs can be selected to engineer TALENs with increased specificity and activity even when co-administered. For example, Gautron disclosed the combined use of T3v4 PD-1 and TRAC TALENs to generate double gene knockout CAR T cells that retain potent anti-tumor functions in vitro.

In some embodiments, TILs can be gene-edited using Gautron's method or other methods described herein, and these TILs can then be amplified by any of the procedures described herein. In one embodiment, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises the steps of: (a) converting the first TILs obtained from the tumor using CD3 and CD28 activating beads or antibodies The population is activated for 1 to 5 days and the tumor is resected from the patient; (b) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs are selected from the first TIL population in (a) to obtain enriched CD39/CD69 a double negative TIL population; (c) gene editing at least a portion of the first TIL population using electroporation of a transcriptional activator effector nuclease to obtain a second TIL population, wherein the gene editing reduces the portion of the second TIL population CD39 and CD69 in the cells; (d) optionally culturing a second population of TILs; (e) performing the first expansion by culturing the second population of TILs in cell culture medium comprising IL-2 and, optionally, OKT-3 (f) by A second expansion is performed by supplementing the cell culture medium of the third TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fourth TIL population, wherein the second expansion is performed for about 7 to 14 day to obtain the fourth TIL population, wherein the fourth TIL population is a therapeutic TIL population; (g) collecting the therapeutic TIL population obtained from step (f); (h) collecting the collected TIL population from step (g) Transfer to an infusion bag, wherein the transfer from steps (f) to (g) is optionally performed without an open system; and (i) optionally, wherein steps (a) to (h) are performed in a closed, sterile system ) one or more.

In some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises the steps of: (a) converting the first TIL obtained from the tumor using CD3 and CD28 activating beads or antibodies The population is activated for 1 to 5 days and the tumor is resected from the patient; (b) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs are selected from the first TIL population in (a) to obtain enriched CD39/CD69 a double negative population of TILs; (c) gene editing at least a portion of the first population of TILs using electroporation of a transcription activator effector nuclease in the cell perforation medium to obtain a second population of TILs, wherein the gene editing reduces the second TILs expression of CD39 and CD69 in the portion of cells in the population; (d) optionally cultivating a second TIL population; (e) by culturing a second TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 A population of TILs is subjected to a first expansion to produce a third population of TILs, wherein the first expansion is performed in a closed vessel providing a first gas-permeable surface area, wherein the first expansion is performed for about 6 to 9 days to obtain a third TIL population TIL population; (f) performing a second expansion by supplementing the cell culture medium of the third TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fourth TIL population, wherein the second The expansion is carried out for about 9 to 11 days to obtain a fourth TIL population, wherein the fourth TIL population is a therapeutic TIL population; (g) collecting the therapeutic TIL population obtained from step (f); (h) collecting the therapeutic TIL population from step (f); ) where the collected TIL population is transferred to an infusion bag, wherein the transfer from steps (f) to (g) is optionally performed without opening the system; and (i) where steps ( one or more of a) to (h).

In some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises the steps of: (a) converting the first TIL obtained from the tumor using CD3 and CD28 activating beads or antibodies The population is activated for 1 to 5 days and the tumor is resected from the patient; (b) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs are selected from the first TIL population in (a) to obtain enriched CD39/CD69 a double negative TIL population; (c) gene editing at least a portion of the first TIL population using electroporation of a transcription activator effector nuclease in the cell perforation medium to obtain a second TIL population, wherein the gene editing reduces the second TIL expression of CD39 and CD69 in the fraction of cells in the population; (d) optionally cultivating a second population of TILs, wherein the culturing is carried out at about 30-40° C. and about 5% CO ₂ ; -2 and optionally OKT-3 cell culture medium for the first expansion of the second TIL population to produce the third TIL population, wherein the first expansion is in a closed vessel providing a first gas permeable surface area proceed, wherein the first expansion is performed for about 6 to 9 days to obtain a third TIL population; (f) by supplementing the cell culture medium of the third TIL population with additional IL-2, OKT-3 and antigen presenting cells (APCs) to perform a second expansion to produce a fourth TIL population, wherein the second expansion is performed for about 9 to 11 days to obtain a fourth TIL population, wherein the fourth TIL population is a therapeutic TIL population; (g) collected from step ( f) The therapeutic TIL population obtained; (h) Transfer of the collected TIL population from step (f) to an infusion bag, wherein the transfer from steps (f) to (g) is optionally without opening the system and (i) optionally wherein one or more of steps (a) to (h) are performed in a closed, sterile system.

According to other embodiments, TALENs can be specifically designed to achieve a higher incidence of DSB events in specifically selected cells of interest capable of targeting genes. See US Patent Publication No. 2013/0315884. The use of such rare cutting endonucleases increases the chance of achieving double inactivation of the gene of interest in transfected cells, enabling the generation of engineered cells, such as T cells. In addition, additional catalytic domains can be introduced with TALENs to increase mutagenesis and enhance target gene inactivation. The TALENs described in US Patent Publication No. 2013/0315884 were successfully used to engineer T cells for immunotherapy. TALENs can also be used to inactivate various immune checkpoint genes in T cells, including at least two genes in a single T cell. See U.S. Patent Publication No. 2016/0120906. In addition, TALENs can be used to inactivate genes encoding targets for immunosuppressants and T cell receptors, as disclosed in US Patent Publication No. 2018/0021379, which is incorporated herein by reference. In addition, TALENs can be used to inhibit the expression of β2-microglobulin (B2M) and/or class II major histocompatibility complex transactivator (CIITA), as disclosed in U.S. Patent Publication No. 2019/0010514 as Incorporated herein by reference.

Non-limiting examples of genes that can be silenced or suppressed by permanent gene editing of TILs using the TALE approach include CD39, CD69, PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

Non-limiting examples of TALE nucleases targeting the PD-1 gene are provided in the table below. In these examples, the gene body sequence of interest contained two 17-base pair (bp) long sequences (called half-targets, shown in uppercase letters) separated by a 15-bp spacer (shown in lowercase letters). Each half-target is recognized by the repeat sequence of the half-TALE-nucleases listed in the table. Thus, according to a particular embodiment, the TALE-nuclease according to the invention recognizes and cleaves a target sequence selected from the group consisting of: SEQ ID NO:238 and SEQ ID NO:239. TALEN sequences and gene editing methods are also described in Gautron discussed above.

In some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises the steps of: (a) converting the first TIL obtained from the tumor using CD3 and CD28 activating beads or antibodies The population is activated for 1 to 5 days and the tumor is resected from the patient; (b) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs are selected from the first TIL population in (a) to obtain enriched CD39/CD69 double negative TIL population; (c) gene editing at least a portion of the first TIL population, wherein the gene editing comprises electroporation in cell perforation medium using transcriptional activator effector nucleases targeting CD39 and CD69 to obtain a second a TIL population, and wherein the gene editing reduces the expression of CD39 and CD69 in the subset of cells in the second TIL population; (d) optionally cultivating the second TIL population, wherein at about 30-40° C. and about 5% CO ₂ culturing; (e) performing a first expansion by culturing a second population of TILs in a cell culture medium comprising IL-2 and, optionally, OKT-3 to produce a third population of TILs, wherein the first expansion is Performed in a closed vessel providing a first gas-permeable surface area, wherein the first expansion is performed for about 6 to 9 days to obtain a third TIL population; (f) by using additional IL-2, OKT-3 and antigen presenting cells (APC) Supplementing the cell culture medium of the third TIL population for a second expansion to produce a fourth TIL population, wherein the second expansion is performed for about 9 to 11 days to obtain the fourth TIL population, wherein the fourth TIL population is therapeutic (g) collecting the therapeutic TIL population obtained from step (f); (h) transferring the collected TIL population from step (f) to an infusion bag, wherein from steps (f) to (g) The transfer is optionally performed without opening the system; and (i) optionally wherein one or more of steps (a) to (h) are performed in a closed, sterile system. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, IL-18, and IL-21.

Other non-limiting examples of genes that can be enhanced by permanent gene editing of TILs via TALE methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1/2 intracellular domain (ICD) and/or NOTCH ligand mDLL1.

Examples of systems, methods, and compositions that alter the expression of target gene sequences by the TALE approach and that can be used in accordance with embodiments of the present invention are described in US Patent No. 8,586,526, which is incorporated herein by reference. Examples of these disclosures include the use of non-naturally occurring DNA-binding polypeptides having two or more TALE-repeat units comprising a repeat RVD, an N-cap polypeptide made from residues of the TALE protein, and C-cap polypeptides made from fragments of the full-length C-terminal region of TALE proteins.

Examples of TALEN design and design strategies, activity assessment, screening strategies and methods that can be used to efficiently perform TALEN-mediated gene integration and inactivation and that can be used in accordance with embodiments of the present invention are described in Valton et al., Methods 》, 2014 , 69, 151-170, which is incorporated herein by reference.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL were selected from the first TIL population in (a), To obtain a CD39/CD69 double-negative TIL population enriched; (d) by culturing the first TIL population in a cell culture medium comprising IL-2 and, optionally, OKT-3 and/or 4-1BB agonist antibodies for about 3 to 11 days to carry out the first expansion to produce the second TIL population, wherein the first expansion is carried out in a closed container providing the first air-permeable surface area; (e) by adding OKT-3 and culturing for about 1 Stimulating the second TIL population for at least 3 days, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) sterile electroporating the second TIL population to achieve transferring at least one gene editor to a plurality of cells in a second population of TILs; (g) resting the second population of TILs for about 1 day; -3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) supplement the cell culture medium of the second TIL population for a second expansion to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days Obtaining a third population of TILs, wherein the second expansion is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) is optionally without opening the system carrying out; (i) collecting the therapeutic TIL population obtained from step (h) to obtain a collected TIL population, wherein the transition from step (h) to step (i) is carried out without opening the system, wherein the The collected TIL population is a therapeutic TIL population; (j) transferring the collected TIL population to an infusion bag, wherein the transfer from steps (i) to (j) is optionally performed without opening the system; and ( k) Cryopreserving the collected TIL populations optionally using cryopreservation media, where the electroporation step comprises delivery of a TALE nuclease system that reduces the expression of CD39 and CD69.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL were selected from the first TIL population in (a), To obtain a CD39/CD69 double-negative TIL population enriched; (d) by culturing the first TIL population in a cell culture medium comprising IL-2 and, optionally, OKT-3 and/or 4-1BB agonist antibodies for about 3 to 11 days to carry out the first expansion to produce the second TIL population, wherein the first expansion is carried out in a closed container providing the first air-permeable surface area; (e) by adding OKT-3 and culturing for about 1 Stimulating the second TIL population for up to 3 days to obtain a second TIL population, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (g) for the second TIL population performing sterile electroporation to effect transfer of at least one gene editor into a plurality of cells in the second TIL population; (h) resting the second TIL population for about 1 day; (i) by treating the second TIL population with additional IL- 2. The OKT-3 antibody selected according to the situation, the OX40 antibody selected according to the situation and the antigen-presenting cell (APC) supplement the cell culture medium of the second TIL population to carry out the second expansion to produce the third TIL population, wherein the second expansion an increase of about 7 to 11 days to obtain a third population of TILs, wherein the second expansion is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (h) to step (i) is optional (j) collecting the third TIL population obtained from step (i) to obtain the collected TIL population, wherein the transition from step (i) to (j) is in the closed system (k) transferring the collected TIL population to an infusion bag, wherein the transfer from steps (j) to (k) is optionally performed without opening the system and (1) optionally cryopreserving the collected TIL population using a cryopreservation medium, wherein the electroporation step comprises delivery of a TALE nuclease system that reduces the expression of CD39 and CD69. c. Zinc fingering

The method for expanding TILs into a therapeutic population can be according to any of the examples of the methods described herein (e.g., Process 2A) or as described in PCT/US2017/058610, PCT/US2018/012605 or PCT/US2018/012633 Performed as described, wherein the method further comprises gene editing at least a portion of the TIL by zinc finger or zinc finger nuclease methods. According to certain embodiments, the use of a zinc finger approach during the TIL expansion process can result in the expression of at least one immunomodulatory composition at the cell surface and, optionally, one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Reticence or reduction in performance. Alternatively, use of a zinc finger approach during the TIL expansion process can result in the expression of at least one immunomodulatory composition at the cell surface and, optionally, enhanced expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population .

Individual zinc fingers in a conserved ββα configuration contain approximately 30 amino acids. Several amino acids on the surface of the α-helix usually contact 3 bp in the DNA major groove with varying levels of selectivity. Zinc fingers have two protein domains. The first domain is the DNA binding domain, which includes eukaryotic transcription factors and contains zinc fingers. The second domain is the nuclease domain, which includes the FokI restriction enzyme and is responsible for catalytic cleavage of DNA.

The DNA binding domain of an individual ZFN typically contains between three and six individual zinc finger repeats and each recognizes between 9 and 18 base pairs. Even a pair of 3-finger ZFNs recognizing a total of 18 base pairs could theoretically target a single locus in the mammalian genome if the zinc finger domains were specific for their intended target sites. One approach to generating new zinc finger arrays is to combine smaller zinc finger "modules" with known specificities. The most common modular assembly process involves combining three separate zinc fingers that each recognize a 3 base pair DNA sequence to generate a 3 finger array that recognizes a 9 base pair target site. Alternatively, selection-based methods, such as oligomerized pool engineering (OPEN), can be used to select new zinc finger arrays from randomly grouped libraries that account for context dependencies between adjacent fingers Sexual interaction (context-dependent interaction). Engineered zinc fingers are commercially available; Sangamo Biosciences (Richmond, CA, USA) has partnered with Sigma-Aldrich (St.Louis, MO, USA) Co-developed a dedicated platform (CompoZr®) for zinc finger construction.

Non-limiting examples of genes that can be silenced or suppressed by permanent gene editing of TILs using the zinc finger approach include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA , CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD , FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

Non-limiting examples of genes that can be enhanced by permanent gene editing of TILs via the zinc finger approach include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1/2 intracellular domain (ICD) and/or NOTCH ligand mDLL1.

Examples of systems, methods, and compositions that alter the expression of target gene sequences by the zinc finger approach and that can be used in accordance with embodiments of the present invention are described in U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136號、第6,824,978號、第6,866,997號、第6,933,113號、第6,979,539號、第7,013,219號、第7,030,215號、第7,220,719號、第7,241,573號、第7,241,574號、第7,585,849號、第7,595,376號、第6,903,185號and No. 6,479,626, which is incorporated herein by reference.

Further examples of systems, methods and compositions that alter the expression of target gene sequences by means of a zinc finger approach and that can be used in accordance with embodiments of the present invention are described in Beane et al., Molecular Therapy, 2015 , 23 1380-1390, Its disclosure is incorporated herein by reference.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL were selected from the first TIL population in (a), To obtain a CD39/CD69 double-negative TIL population enriched; (d) by culturing the first TIL population in a cell culture medium comprising IL-2 and, optionally, OKT-3 and/or 4-1BB agonist antibodies for about 3 to 11 days to carry out the first expansion to produce the second TIL population, wherein the first expansion is carried out in a closed container providing the first air-permeable surface area; (e) by adding OKT-3 and culturing for about 1 Stimulating the second TIL population for up to 3 days to obtain a second TIL population, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) for the second TIL population performing sterile electroporation to effect transfer of at least one gene editor into a plurality of cells in the second population of TILs; (g) resting the second population of TILs for about 1 day; 2. The OKT-3 antibody selected according to the situation, the OX40 antibody selected according to the situation and the antigen-presenting cell (APC) supplement the cell culture medium of the second TIL population to carry out the second expansion to produce the third TIL population, wherein the second expansion The increase is carried out for about 7 to 11 days to obtain a third TIL population, wherein the second expansion is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (g) to step (h) is performed at carried out with an open system; (i) collecting the therapeutic TIL population obtained from step (h) to obtain a collected TIL population, wherein the transition from step (h) to step (i) is without an open system wherein the collected TIL population is a therapeutic TIL population; (j) transferring the collected TIL population to an infusion bag, wherein the transfer from steps (i) to (j) is optionally performed without opening the system and (k) optionally cryopreserving the collected TIL population using a cryopreservation medium, wherein the electroporation step comprises delivery of a zinc finger nuclease system that reduces the expression of CD39 and CD69.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL were selected from the first TIL population in (a), To obtain a CD39/CD69 double-negative TIL population enriched; (d) by culturing the first TIL population in a cell culture medium comprising IL-2 and, optionally, OKT-3 and/or 4-1BB agonist antibodies for about 3 to 11 days to carry out the first expansion to produce the second TIL population, wherein the first expansion is carried out in a closed container providing the first air-permeable surface area; (e) by adding OKT-3 and culturing for about 1 Stimulating the second TIL population for at least 3 days, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) sterile electroporating the second TIL population to achieve transferring at least one gene editor to a plurality of cells in a second population of TILs; (g) resting the second population of TILs for about 1 day; -3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) supplement the cell culture medium of the second TIL population for a second expansion to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days obtaining a third TIL population, wherein the second expansion is performed in a closed container providing a second gas permeable surface area, and wherein the transition from step (g) to step (h) is performed without opening the system; (i) collecting the therapeutic TIL population obtained from step (h) to obtain a collected TIL population, wherein the transition from step (h) to step (i) is performed without opening the system, wherein the collected The TIL population is a therapeutic TIL population; (j) transferring the collected TIL population to an infusion bag, wherein the transfer from steps (i) to (j) is optionally performed without opening the system; and (k) Collected TIL populations are optionally cryopreserved using cryopreservation media, where the electroporation step involves delivery of a zinc finger nuclease system that reduces the expression of CD39 and CD69. D. Immune Checkpoints

According to certain embodiments of the present invention, the TIL population is gene-edited to express one or more immunomodulatory compositions and one or more immune checkpoint genes in the genetically modified TIL population on the cell surface of TIL cells in the TIL population. In other words, in addition to the modification of the TIL population for expression of one or more immunomodulatory compositions on the cell surface, the DNA sequence within the TIL encoding one or more immune checkpoints of the TIL is permanently modified in the gene body of the TIL, For example insertions, deletions or substitutions. Immune checkpoints are molecules expressed by lymphocytes that modulate immune responses through inhibitory or stimulatory pathways. In the case of cancer, immune checkpoint pathways are often activated to suppress anti-tumor responses, ie, expression of certain immune checkpoints by malignant cells suppresses anti-tumor immunity and favors cancer cell growth. See, eg, Marin-Acevedo et al., J Hematology & Oncology (2018) 11:39. Accordingly, certain inhibitory checkpoint molecules serve as targets for immunotherapy of the present invention. According to certain embodiments, TILs are gene-edited to block or stimulate certain immune checkpoint pathways and thereby enhance the body's immune activity against tumors.

As used herein, an immune checkpoint gene comprises a DNA sequence encoding an immune checkpoint molecule. According to certain embodiments of the invention, gene editing of TILs during the TIL expansion method results in silencing or reduction of expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. For example, gene editing can result in silencing or reducing the expression of inhibitory receptors such as PD-1 or CTLA-4, thereby enhancing the immune response.

The most widely studied checkpoints include programmed cell death receptor-1 (PD-1) and cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4), which are inhibitory receptors on immune cells that, when combined with Inhibitory ligands inhibit important effector functions (eg, activation, proliferation, cytokine release, cytotoxicity, etc.) upon interaction. In addition to PD-1 and CTLA-4, a number of checkpoint molecules have emerged as potential targets for immunotherapy, as discussed in more detail below.

Non-limiting examples of immune checkpoint genes that can be silenced or suppressed by the permanent gene editing TIL of the present invention include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL -B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF(BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3. For example, immune checkpoint genes that can be silenced or suppressed in TILs of the invention can be selected from the group comprising: PD-1, CTLA-4, LAG-3, TIM-3, Cish, TGFβ, and PKA. BAFF(BR3) is described in Bloom et al., J. Immunother. , 2018 , in press. According to another example, immune checkpoint genes that can be silenced or suppressed in TILs of the invention can be selected from the group comprising: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3) and combinations thereof.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL were selected from the first TIL population in (a), To obtain a CD39/CD69 double-negative TIL population enriched; (d) by culturing the first TIL population in a cell culture medium comprising IL-2 and, optionally, OKT-3 and/or 4-1BB agonist antibodies for about 3 to 11 days to carry out the first expansion to produce the second TIL population, wherein the first expansion is carried out in a closed container providing the first air-permeable surface area; (e) by adding OKT-3 and culturing for about 1 Stimulating the second TIL population for at least 3 days, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) sterile electroporating the second TIL population to achieve transferring at least one gene editor to a plurality of cells in a second population of TILs; (g) resting the second population of TILs for about 1 day; -3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) supplement the cell culture medium of the second TIL population for a second expansion to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days , wherein the second amplification is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (g) to step (h) is optionally performed without opening the system; (i) collecting The third TIL population obtained from step (h) to obtain the collected TIL population, wherein the transition from step (h) to step (i) is optionally carried out without opening the system, wherein the collected TIL population being a therapeutic TIL population; (j) transferring the collected TIL population to an infusion bag, wherein the transfer from steps (i) to (j) is optionally performed without opening the system; and (k) optionally The collected TIL population is cryopreserved using a cryopreservation medium, wherein the electroporation step comprises delivery of at least one gene editor system selected from the group consisting of: Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, transcriptional activator A TALE-like effector (TALE) system or zinc finger system, wherein the at least one gene editor system reduces the expression of CD39 and CD69. 1. PD-1

One of the most studied targets for inducing checkpoint blockade is the programmed death receptor (PD1 or PD-1, also known as PDCD1), a member of the CD28 superfamily of T cell regulators. Its ligands PD-L1 and PD-L2 are expressed on various tumor cells, including melanoma. The interaction of PD-1 and PD-L1 can inhibit T cell effector function, cause T cell depletion in chronic stimulation environment and induce T cell apoptosis in tumor microenvironment. PD1 may also play a role in tumor-specific evasion of immune surveillance.

According to certain embodiments, the compositions and methods according to the invention silence or reduce the expression of PD1 in TILs. For example, expansion of tumor infiltrating lymphocytes (TILs) into therapeutic A method for a population of TILs, wherein the method comprises gene editing at least a portion of TILs by silencing or inhibiting the expression of PD1. As described in more detail below, the gene editing process can involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at immune checkpoint genes, such as PD1. For example, the expression of PD1 in TILs can be silenced or reduced using CRISPR methods, TALE methods or zinc finger methods. 2. CTLA-4

CTLA-4 expression is induced upon T cell activation on activated T cells and competes for binding to antigen presenting cell activating antigens CD80 and CD86. The interaction of CTLA-4 with CD80 or CD86 can cause T cell suppression and serve to maintain the balance of the immune response. However, inhibiting the interaction of CTLA-4 with CD80 or CD86 can prolong T cell activation and thus increase the level of immune responses against cancer antigens.

According to certain embodiments, the compositions and methods according to the invention silence or reduce the expression of CTLA-4 in TILs. For example, expansion of tumor infiltrating lymphocytes (TILs) can be performed according to any of the embodiments of the methods described herein (e.g., Procedure 2A, Procedure Gen 3, or the methods shown in Figures 34 and 35). A method of augmenting a population of therapeutic TILs, wherein the method comprises gene editing at least a portion of the TILs to express at least one immunomodulatory composition on the cell surface and silencing or inhibiting expression of CTLA-4 in the TILs. As described in more detail below, the gene editing process may involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at immune checkpoint genes, such as CTLA-4. For example, CTLA-4 expression in TILs can be silenced or inhibited using CRISPR approach, TALE approach or zinc finger approach. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, and IL-21. 3. LAG-3

Lymphocyte activation gene-3 (LAG-3, CD223) is expressed by T cells and natural killer (NK) cells following class II major histocompatibility complex (MHC) engagement. Although the mechanism is not clear, its regulation can lead to negative regulation of T cell function, preventing tissue damage and autoimmunity. LAG-3 and PD-1 are often co-expressed and upregulated on TILs, leading to immune exhaustion and tumor growth. Thus, LAG-3 blockade may improve antitumor responses. See, eg, Marin-Acevedo et al., J Hematology & Oncology (2018) 11:39.

According to certain embodiments, the compositions and methods according to the invention silence or reduce the expression of LAG-3 in TILs. For example, expansion of tumor infiltrating lymphocytes (TILs) can be performed according to any of the embodiments of the methods described herein (e.g., Procedure 2A, Procedure Gen 3, or the methods shown in Figures 34 and 35). A method of augmenting a population of therapeutic TILs, wherein the method comprises gene editing at least a portion of the TILs to express at least one immunomodulatory composition on the cell surface and silencing or inhibiting expression of LAG-3 in the TILs. As described in more detail below, the gene editing process may involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at immune checkpoint genes such as LAG-3. According to certain embodiments, the expression of LAG-3 in TILs can be silenced or inhibited using CRISPR method, TALE method or zinc finger method. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, and IL-21. 4. TIM-3

T cell immunoglobulin-3 (TIM-3) is a direct negative regulator of T cells and is expressed on NK cells and macrophages. TIM-3 indirectly contributes to immunosuppression by inducing the expansion of myeloid-derived suppressor cells (MDSCs). It was found to be specifically elevated on dysfunctional and exhausted T cells, suggesting an important role in malignant disease.

According to certain embodiments, the compositions and methods according to the invention silence or reduce the expression of TIM-3 in TILs. For example, expansion of tumor infiltrating lymphocytes (TILs) can be performed according to any of the embodiments of the methods described herein (e.g., Procedure 2A, Procedure Gen 3, or the methods shown in Figures 34 and 35). A method of augmenting a population of therapeutic TILs, wherein the method comprises gene editing at least a portion of the TILs to express at least one immunomodulatory composition on the cell surface and silencing or inhibiting expression of TIM-3 in the TILs. As described in more detail below, the gene editing process can involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at immune checkpoint genes, such as TIM-3. For example, the expression of TIM-3 in TILs can be silenced or inhibited using CRISPR approach, TALE approach or zinc finger approach. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, and IL-21. 5. Cish

Cish, a member of the suppressor of interleukin signaling (SOCS) family, is induced by TCR stimulation in CD8+ T cells and inhibits their functional affinity for tumors. Genetic deletion of Cish in CD8+ T cells enhances their expansion, functional avidity, and cytokine multifunctionality, leading to marked and durable regression of existing tumors. See, eg, Palmer et al., Journal of Experimental Medicine , 212(12): 2095 (2015).

According to certain embodiments, the compositions and methods according to the invention silence or reduce the expression of Cish in TILs. For example, expansion of tumor infiltrating lymphocytes (TILs) can be performed according to any of the embodiments of the methods described herein (e.g., Procedure 2A, Procedure Gen 3, or the methods shown in Figures 34 and 35). A method of augmenting a population of therapeutic TILs, wherein the method comprises gene editing at least a portion of the TILs to express at least one immunomodulatory composition on the cell surface and silencing or inhibiting expression of Cish in the TILs. As described in more detail below, the gene editing process may involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at immune checkpoint genes, such as Cish. For example, the expression of Cish in TILs can be silenced or inhibited using CRISPR approach, TALE approach or zinc finger approach. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, and IL-21. 6. TGFβ

The TGFβ signaling pathway has multiple functions in regulating cell growth, differentiation, apoptosis, motility and invasion, extracellular matrix production, angiogenesis and immune response. Dysregulation of TGFβ signaling is common in tumors and plays an important role in tumor initiation, progression and metastasis. At the microenvironmental level, the TGFβ pathway contributes to the creation of a microenvironment conducive to tumor growth and metastasis in all carcinogenesis. See, eg, Neuzillet et al., Pharmacology & Therapeutics, Vol. 147, pp. 22-31 (2015).

According to certain embodiments, the compositions and methods according to the invention silence or reduce the expression of TGF[beta] in TILs. For example, expansion of tumor infiltrating lymphocytes (TILs) can be performed according to any of the embodiments of the methods described herein (e.g., Procedure 2A, Procedure Gen 3, or the methods shown in Figures 34 and 35). A method of augmenting a population of therapeutic TILs, wherein the method comprises gene editing at least a portion of the TILs to express at least one immunomodulatory composition on the cell surface and silencing or inhibiting expression of TGFβ in the TILs. As described in more detail below, the gene editing process may involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at immune checkpoint genes such as TGFβ. For example, the expression of TGFβ in TILs can be silenced or inhibited using CRISPR approach, TALE approach or zinc finger approach. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, and IL-21.

In some embodiments, TGFβR2 (TGFβ receptor 2) can be inhibited by silencing TGFβR2 using the CRISPR/Cas9 system or by using a TGFβR2 dominant-negative extracellular trap using methods known in the art. 7. PKA

Protein kinase A (PKA) is a member of the well-known serine-threonine protein kinase superfamily. PKA, also known as cAMP-dependent protein kinase, is a multiunit protein kinase that mediates signal transduction of G protein-coupled receptors through its activation upon cAMP binding. It is involved in the control of a variety of cellular processes from metabolism to ion channel activation, cell growth and differentiation, gene expression and apoptosis. Importantly, PKA is involved in the initiation and progression of many tumors. See eg Sapio et al., EXCLI Journal ; 2014; 13: 843-855.

According to certain embodiments, the compositions and methods according to the invention silence or reduce the expression of PKA in TILs. For example, expansion of tumor infiltrating lymphocytes (TILs) can be performed according to any of the embodiments of the methods described herein (e.g., Procedure 2A, Procedure Gen 3, or the methods shown in Figures 34 and 35). A method of augmenting a population of therapeutic TILs, wherein the method comprises gene editing at least a portion of the TILs to express at least one immunomodulatory composition on the cell surface and silencing or inhibiting expression of PKA in the TILs. As described in more detail below, the gene editing process can involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at immune checkpoint genes, such as PKA. For example, the expression of PKA in TILs can be silenced or inhibited using CRISPR approach, TALE approach or zinc finger approach. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, and IL-21. 8. CBLB

CBLB (or CBL-B) is an E3 ubiquitin-protein ligase and a negative regulator of T cell activation. Bachmaier et al., Nature , 2000, 403, 211-216; Wallner et al., Clin. Dev. Immunol. 2012, 692639.

According to certain embodiments, the compositions and methods according to the invention silence or reduce the expression of CBLB in TILs. For example, expansion of tumor infiltrating lymphocytes (TILs) can be performed according to any of the embodiments of the methods described herein (e.g., Procedure 2A, Procedure Gen 3, or the methods shown in FIGS. 34 and 35 ). A method of augmenting a population of therapeutic TILs, wherein the method comprises gene editing at least a portion of the TILs to express at least one immunomodulatory composition on the cell surface and silencing or inhibiting expression of CBLB in the TILs. As described in more detail below, the gene editing process may involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at immune checkpoint genes such as CBLB. For example, the expression of PKA in TILs can be silenced or inhibited using CRISPR approach, TALE approach or zinc finger approach. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, and IL-21. In some embodiments, CBLB is silenced using TALEN knockout. In some embodiments, CBLB is silenced using TALE-KRAB transcriptional inhibitor gene insertion. More details on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. 9. TIGIT

T cell immunoreceptors with Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains or TIGIT are transmembrane glycoprotein receptors that have an Ig-like V-type domain and ITIM in their cytoplasmic domain. Khalil et al., Advances in Cancer Research, 2015 , 128, 1-68; Yu et al., Nature Immunology, 2009 , Vol. 10, No. 1, 48- 57. TIGIT is expressed by some T cells and natural killer cells. Furthermore, TIGIT has been shown to be overexpressed on antigen-specific CD8+ T cells and CD8+ TILs, especially from individuals with melanoma. Studies have demonstrated that the TIGIT pathway contributes to tumor immune evasion and that TIGIT inhibition has been shown to increase T cell activation and proliferation in response to polyclonal and antigen-specific stimuli. Khalil et al., Advances in Cancer Research, 2015 , 128, 1-68. Furthermore, co-blockade of TIGIT with PD-1 or TIM3 demonstrated synergy against solid tumors in mouse models. Ibid; see also Kurtulus et al., The Journal of Clinical Investigation, 2015 , Vol. 125, No. 11, 4053-4062.

According to certain embodiments, the compositions and methods according to the invention silence or reduce the expression of TIGIT in TILs. For example, expansion of tumor infiltrating lymphocytes (TILs) can be performed according to any of the embodiments of the methods described herein (e.g., Procedure 2A, Procedure Gen 3, or the methods shown in Figures 34 and 35). A method of augmenting a population of therapeutic TILs, wherein the method comprises gene editing at least a portion of the TILs to express at least one immunomodulatory composition on the cell surface and silencing or inhibiting expression of TIGIT in the TILs. As described in more detail below, the gene editing process may involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at immune checkpoint genes such as TIGIT. For example, the expression of TIGIT in TILs can be silenced or inhibited using CRISPR approach, TALE approach or zinc finger approach. In some embodiments, at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, and IL-21. 10. Poison

Thymocyte selection-associated high mobility group (HMG) cassette (TOX) is a transcription factor that contains the HMG cassette DNA-binding domain. TOX, a member of the HMG cassette superfamily, is thought to bind DNA in a sequence-independent, but structure-dependent manner.

TOX has been identified as an important regulator of tumor-specific CD8 ⁺ T cell dysfunction or T cell depletion and found to program CD8 ⁺ T cell depletion both transcriptionally and epigenetically, as e.g. Scott et al., Nature , 2019 , 571, 270-274 and described in Khan et al., Nature, 2019 , 571, 211-218, which are incorporated herein by reference in their entirety. TOX was also found to be an important factor in the development and maintenance of depleted T cells during chronic infection, as described in Alfei et al., Nature, 2019 , 571, 265-269, which is incorporated by reference in its entirety into this article. TOX is abundantly expressed in dysfunctional or depleted T cells from tumors and chronic viral infections. Ectopic expression of TOX in effector T cells in vitro induces a transcriptional program associated with T cell depletion, whereas deletion of TOX in T cells abolishes the T depletion program.

According to certain embodiments, the compositions and methods according to the invention silence or reduce the expression of TOX in TIL. For example, expansion of tumor infiltrating lymphocytes (TILs) can be performed according to any of the embodiments of the methods described herein (e.g., Procedure 2A, Procedure Gen 3, or the methods shown in Figures 34 and 35). A method of augmenting a population of therapeutic TILs, wherein the method comprises gene editing at least a portion of the TILs to express at least one immunomodulatory composition and silence or inhibit expression of TOX on the cell surface. As described in more detail below, the gene editing process may involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at immune checkpoint genes such as TOX. For example, the expression of TOX in TILs can be silenced or inhibited using CRISPR approach, TALE approach or zinc finger approach. In some embodiments, the at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, and IL-21. E. Overexpression of co-stimulatory receptors or adhesion molecules

According to other embodiments, gene editing of TILs during the TIL expansion method can result in the expression of at least one immunomodulatory composition at the cell surface and one or more costimulatory receptors, adhesion molecules, and TILs in at least a portion of the therapeutic TIL population. / or enhanced expression of cytokines. For example, gene editing can result in enhanced expression of costimulatory receptors, adhesion molecules or cytokines, ie, overexpression compared to expression of costimulatory receptors, adhesion molecules or cytokines that have not been genetically modified. Non-limiting examples of co-stimulatory receptor, adhesion molecule, or interleukin genes that may exhibit enhanced expression by the permanently gene-edited TILs of the invention include certain chemokine receptors and interleukins, such as CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1/2 intracellular domain (ICD) and/or NOTCH Ligand mDLL1. 1. CCR

For recipient T cell immunotherapy to be effective, proper migration of T cells into tumors by chemoattractants is required. Matching between chemoattractants secreted by tumor cells, chemoattractants present in the periphery, and chemoattractant receptors expressed by T cells is important for the successful migration of T cells into the tumor bed.

According to certain embodiments, the gene editing method of the present invention can be used to increase the expression of certain chemokine receptors (such as one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3 and CX3CR1) in TIL. CCR overexpression may contribute to the promotion of effector function and proliferation of TILs following recipient transfer.

According to certain embodiments, the compositions and methods according to the invention enhance the expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and CX3CR1 in TILs. For example, expansion of tumor infiltrating lymphocytes (TILs) can be performed according to any of the embodiments of the methods described herein (e.g., Procedure 2A, Procedure Gen 3, or the methods shown in Figures 34 and 35). A method of augmenting a population of therapeutic TILs, wherein the method comprises gene editing at least a portion of the TILs to express at least one immunomodulatory composition on the cell surface and enhancing one of CCR2, CCR4, CCR5, CXCR2, CXCR3, and CX3CR1 in the TILs or The performance of many. In some embodiments, the at least one immunomodulatory composition comprises a cytokine fused to a membrane anchor. In some embodiments, the interleukin is selected from the group consisting of IL-12, IL-15, IL-18, and IL-21.

As described in more detail below, the gene editing process may involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at the chemokine receptor gene. For example, the expression of certain chemokine receptors in TILs can be enhanced using CRISPR approach, TALE approach or zinc finger approach.

In some embodiments, CCR4 and/or CCR5 adhesion molecules are inserted into the TIL population using γ-retroviral or lentiviral methods as described herein. In one embodiment, the CXCR2 adhesion molecule is inserted into the TIL population using a gamma-retroviral or lentiviral approach as described in: Forget et al., Frontiers Immunology 2017 , 8 , 908 or Peng et al., Clin. 2010 , 16 , 5458, the disclosures of which are incorporated herein by reference.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL were selected from the first TIL population in (a), To obtain a CD39/CD69 double-negative TIL population enriched; (d) by culturing the first TIL population in a cell culture medium comprising IL-2 and, optionally, OKT-3 and/or 4-1BB agonist antibodies for about 3 to 11 days to carry out the first expansion to produce the second TIL population, wherein the first expansion is carried out in a closed container providing the first air-permeable surface area; (e) by adding OKT-3 and culturing for about 1 Stimulating the second TIL population for at least 3 days to obtain a second TIL population, wherein the number of the second TIL population is at least 50 times greater than the first TIL population, and wherein the transition from step (d) to step (e) depends on The case is carried out without opening the system; (f) aseptically electroporating the second TIL population to achieve transfer of at least one gene editor into the plurality of cells in the second TIL population; (g) the second TIL population The TIL population is rested for approximately 1 day; (h) by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) to perform a second amplification to produce a third population of TILs, wherein the second amplification is performed for about 7 to 11 days, wherein the second amplification is performed in a closed vessel providing a second gas-permeable surface area, and wherein the g) the transition to step (h) is carried out optionally without opening the system; (i) collecting the third TIL population obtained from step (h) to obtain the collected TIL population, wherein from step (h) The transition to step (i) is optionally performed without opening the system, wherein the collected TIL population is a therapeutic TIL population; (j) transferring the collected TIL population to an infusion bag, wherein from step (i) ) to (j) is optionally performed without opening the system; and (k) optionally using a cryopreservation medium to cryopreserve the collected TIL population, wherein the electroporation step comprises delivery of at least one selected from the group consisting of A group of gene editor systems: clustered regularly interspaced short palindromic repeats (CRISPR) system, transcription activator-like effector (TALE) system or zinc finger system, wherein the at least one gene editor system reduces the difference between CD39 and CD69 expression, and further wherein the at least one gene editor system enables expression of the CXCR2 adhesion molecule at the cell surface of a plurality of cells of the second TIL population, or adhesion of CXCR2 by gamma retroviral or lentiviral methods The molecule inserts into the first TIL population, the second TIL population, or the collected TIL population.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL were selected from the first TIL population in (a), To obtain a CD39/CD69 double-negative TIL population enriched; (d) by culturing the first TIL population in a cell culture medium comprising IL-2 and, optionally, OKT-3 and/or 4-1BB agonist antibodies for about 3 to 11 days to carry out the first expansion to produce the second TIL population, wherein the first expansion is carried out in a closed container providing the first air-permeable surface area; (e) by adding OKT-3 and culturing for about 1 Stimulating the second TIL population for at least 3 days, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) sterile electroporating the second TIL population to achieve transferring at least one gene editor to a plurality of cells in a second population of TILs; (g) resting the second population of TILs for about 1 day; -3 antibody, optionally OX40 antibody, and antigen presenting cells (APCs) supplement the cell culture medium of the second TIL population for a second expansion to generate a third TIL population, wherein the second expansion is performed for about 7 to 11 days , wherein the second amplification is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (g) to step (h) is optionally performed without opening the system; (i) collecting The third TIL population obtained from step (h) to obtain the collected TIL population, wherein the transition from step (h) to step (i) is optionally carried out without opening the system, wherein the collected TIL population being a therapeutic TIL population; (j) transferring the collected TIL population to an infusion bag, wherein the transfer from steps (i) to (j) is optionally performed without opening the system; and (k) optionally The collected TIL population is cryopreserved using a cryopreservation medium, wherein the electroporation step comprises delivery of at least one gene editor system selected from the group consisting of: Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, transcriptional activator Effector-like (TALE) system or zinc finger system, wherein the at least one gene editor system reduces the expression of CD39 and CD69 and further wherein the at least one gene editor system achieves expression at the cell surface of a plurality of cells of the second TIL population Expression of CCR4 and/or CCR5 adhesion molecules, or insertion of CXCR2 adhesion molecules into the first TIL population, the second TIL population, or the collected TIL population by γ-retroviral or lentiviral methods.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL were selected from the first TIL population in (a), To obtain a CD39/CD69 double-negative TIL population enriched; (d) by culturing the first TIL population in a cell culture medium comprising IL-2 and, optionally, OKT-3 and/or 4-1BB agonist antibodies for about 3 to 11 days to carry out the first expansion to produce the second TIL population, wherein the first expansion is carried out in a closed container providing the first air-permeable surface area; (e) by adding OKT-3 and culturing for about 1 Stimulating the second TIL population for up to 3 days to obtain a second TIL population, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) for the second TIL population performing sterile electroporation to effect transfer of at least one gene editor into a plurality of cells in the second population of TILs; (g) resting the second population of TILs for about 1 day; 2. The OKT-3 antibody selected according to the situation, the OX40 antibody selected according to the situation and the antigen-presenting cell (APC) supplement the cell culture medium of the second TIL population to carry out the second expansion to produce the third TIL population, wherein the second expansion The amplification is carried out for about 7 to 11 days, wherein the second amplification is carried out in a closed container providing a second air-permeable surface area, and wherein the transition from step (g) to step (h) is optionally without opening the system (i) collecting the third TIL population obtained from step (h) to obtain the collected TIL population, wherein the transition from step (h) to step (i) is optionally carried out without opening the system , wherein the collected TIL population is a therapeutic TIL population; (j) transferring the collected TIL population to an infusion bag, wherein the transfer from steps (i) to (j) is optionally performed without opening the system and (k) optionally cryopreserving the collected TIL population using a cryopreservation medium, wherein the electroporation step comprises delivering at least one gene editor system selected from the group consisting of: clustered regularly interspaced short palindromic repeats ( CRISPR) system, transcription activator-like effector (TALE) system or zinc finger system, wherein the at least one gene editor system reduces the expression of CD39 and CD69, and further wherein the at least one gene editor system achieves expression of a second TIL population Expression of an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof at the cell surface of a plurality of cells, or by gamma retroviruses A viral or lentiviral approach inserts the adhesion molecule into the first TIL population, the second TIL population, or the collected TIL population. 2. Interleukin

According to other embodiments, the gene editing method of the present invention can be used to increase certain interleukins, such as IL-2, IL-4, IL-7, IL-10, IL-15, IL-18 and IL-21 one or more). Certain interleukins have been shown to enhance T cell effector functions and mediate tumor control.

According to certain embodiments, one or more of IL-2, IL-4, IL-7, IL-10, IL-15, IL-18 and IL-21 in TILs are enhanced according to the compositions and methods of the present invention performance. For example, expansion of tumor infiltrating lymphocytes (TILs) can be performed according to any of the embodiments of the methods described herein (e.g., Procedure 2A, Procedure Gen 3, or the methods shown in Figures 34 and 35). A method of increasing a therapeutic TIL population, wherein the method comprises enhancing one or more of IL-2, IL-4, IL-7, IL-10, IL-15, IL-18 and IL-21 performance to gene edit at least a portion of the TIL. As described in more detail below, the gene editing process may involve the use of programmable nucleases that mediate the generation of double- or single-stranded breaks at the interleukin gene. For example, the expression of certain interleukins in TILs can be enhanced using the CRISPR approach, the TALE approach, or the zinc finger approach.

According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a tumor sample obtained from a patient by processing the tumor sample into a plurality of tumor fragments; The first TIL population was obtained from the resected tumor; (b) tumor fragments were added to the closed system; (c) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL were selected from the first TIL population in (a), To obtain a CD39/CD69 double-negative TIL population enriched; (d) by culturing the first TIL population in a cell culture medium comprising IL-2 and, optionally, OKT-3 and/or 4-1BB agonist antibodies for about 3 to 11 days to carry out the first expansion to produce the second TIL population, wherein the first expansion is carried out in a closed container providing the first air-permeable surface area; (e) by adding OKT-3 and culturing for about 1 Stimulating the second TIL population for up to 3 days to obtain a second TIL population, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) for the second TIL population performing sterile electroporation to effect transfer of at least one gene editor; (g) resting the second TIL population in the plurality of cells in the second TIL population for about 1 day; 2. The OKT-3 antibody selected according to the situation, the OX40 antibody selected according to the situation and the antigen-presenting cell (APC) supplement the cell culture medium of the second TIL population to carry out the second expansion to produce the third TIL population, wherein the second expansion The amplification is carried out for about 7 to 11 days, wherein the second amplification is carried out in a closed container providing a second air-permeable surface area, and wherein the transition from step (g) to step (h) is optionally without opening the system (i) collecting the therapeutic TIL population obtained from step (h) to obtain a collected TIL population, wherein the transition from step (h) to step (i) is carried out without opening the system, wherein The collected TIL population is a therapeutic TIL population; (j) transferring the collected TIL population to an infusion bag, wherein the transfer from steps (i) to (j) is optionally performed without opening the system; and (k) optionally cryopreserving the collected TIL population using a cryopreservation medium, wherein the electroporation step comprises delivery of at least one gene editor system selected from the group consisting of: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a transcription activator-like effector (TALE) system or a zinc finger system, wherein the at least one gene editor system reduces the expression of CD39 and CD69 and further wherein the at least one gene editor system achieves a plurality of cells of a second TIL population Expression of an interleukin selected from the group consisting of IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, and combinations thereof at the cell surface, or by gamma reverse transcription Viral or lentiviral methods insert interleukins into the first TIL population, the second TIL population, or the collected TIL population. D. Protein kinase B (AKT) inhibitors

According to some embodiments, the first amplification step, the second amplification or the first and second amplification steps comprise adding a protein kinase B (AKT) inhibitor (AKTi) to the culture medium. According to some embodiments, initiating the first amplification step, the rapid second amplification or initiating the first and rapid second amplification steps comprises adding a protein kinase B (AKT) inhibitor (AKTi) to the culture medium.

AKT inhibitor

SB-203580

In one embodiment, the AKT inhibitor is SB-203580. The chemical structure and name of SB-203580 are shown as: 4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine

。

.

MK-2206

In one embodiment, the AKT inhibitor is MK-2206. The chemical structure and name of MK-2206 are shown as: 8-[4-(1-Aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4 -f][1,6]naphthyridin-3-one

。

.

SC79

In one embodiment, the AKT inhibitor is SC79. The chemical structure and name of SC79 are shown as: 2-amino-6-chloro-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-benzopyran-3- ethyl formate

。

.

Capivasertib (AZD5363)

In one embodiment, the AKT inhibitor is calvatinib. The chemical structure and name of calvatinib are shown as: 4-amino-N-[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-(7H-pyrrolo[2 ,3-d]pyrimidin-4-yl)piperidine-4-carboxamide

。

.

Miltefosine

In one embodiment, the AKT inhibitor is miltefosine. The chemical structure and name of miltefosine are shown as: 2-(trimethylammonio) ethyl hexadecyl phosphate

。

.

Perifosine

In one embodiment, the AKT inhibitor is perifosine. The chemical structure and name of Perifosin are shown as: (1,1-dimethylpiperidin-1-ium-4-yl)octadecyl phosphate

。

.

PF-04691502

In one embodiment, the AKT inhibitor is PF-04691502. The chemical structure and name of PF-04691502 are shown as: 2-Amino-8-[4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4- Pyrido[2,3-d]pyrimidin-7-one

。

.

CCT128930

In one embodiment, the AKT inhibitor is CCT128930. The chemical structure and name of CCT128930 are shown as: 4-[(4-chlorophenyl)methyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine

。

.

A-674563

In one embodiment, the AKT inhibitor is A-674563. The chemical structure and name of A-674563 are shown as: (2S)-1-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxy-3-phenylpropane- 2-amine

。

.

Archexin (RX-0201)

In one embodiment, the AKT inhibitor is azithromycin. In one embodiment, the AKT inhibitor is an oligodeoxynucleotide having the sequence 5' gctgcatgatctccttggcg 3'.

Oleandrin (PBI-05204)

In one embodiment, the AKT inhibitor is oleandrin. The chemical structure and name of oleandrin are shown as: [(3S,5R,8R,9S,10S,13R,14S,16S,17R)-14-hydroxy-3-[(2R,4S,5S,6S)-5- Hydroxy-4-methoxy-6-methyloxan-2-yl]oxy-10,13-dimethyl-17-(5-oxo-2H-furan-3-yl)- 1,2,3,4,5,6,7,8,9,11, 12,15,16,17-Tetrahydrocyclopenta[a]phenanthren-16-yl]acetate

。

.

AKT inhibitor VIII

In one embodiment, the AKT inhibitor is AKT inhibitor VIII. The chemical structure and name of AKT inhibitor VIII are shown as: 3-[1-[[4-(7-phenyl-3H-imidazo[4,5-g]quinoxolin-6-yl)phenyl]methano Base] piperidin-4-yl] -1H-benzimidazol-2-one

。

.

AT7867

In one embodiment, the AKT inhibitor is AT7867. The chemical structure and name of AT7867 are shown as: 4-(4-Chlorophenyl)-4-[4-(1H-pyrazol-4-yl)phenyl]piperidine

。

.

AT13148

In one embodiment, the AKT inhibitor is AT13148. The chemical structure and name of AT13148 are shown as: (1S)-2-amino-1-(4-chlorophenyl)-1-[4-(1H-pyrazol-4-yl)phenyl]ethanol

。

.

Ipatasertib (GDC-0068)

-1-基)-3-(異丙胺基)丙-1-酮 In one embodiment, the AKT inhibitor is pataxerti. The chemical structure and name of pataxerti are shown as: (S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7 -Dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piper

-1-yl)-3-(isopropylamino)propan-1-one

。

.

TIC10

In one embodiment, the AKT inhibitor is TIC10. The chemical structure and name of TIC10 are shown as: 7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a] Pyrido[3,4-e]pyrimidin-5(4H)-one

。

.

SC79

In one embodiment, the AKT inhibitor is SC79. The chemical structure and name of SC79 are shown as: 2-amino-6-chloro-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-benzopyran-3- ethyl formate

。

.

GSK690693

二唑-3-基)-1-乙基--7-[[(3S)-哌啶-3-基]甲氧基]咪唑并[4,5-c]吡啶-4-基]-2-甲基丁-3-炔-2-醇 In one embodiment, the AKT inhibitor is GSK690693. The chemical structure and name of GSK690693 are shown as: 4-[2-(4-amino-1,2,5-

Oxadiazol-3-yl)-1-ethyl--7-[[(3S)-piperidin-3-yl]methoxy]imidazo[4,5-c]pyridin-4-yl]-2 -Methylbut-3-yn-2-ol

。

.

Afuresertib (GSK2110183)

In one embodiment, the AKT inhibitor is afetinib. The chemical structure and name of afotinib are shown as: N-[(2S)-1-amino-3-(3,4-difluorophenyl)propan-2-yl]-5-chloro-4-( 4-chloro-2-methylpyrazol-3-yl)thiophene-2-carboxamide.

。

.

Urosertib (GSK2141795)

In one embodiment, the AKT inhibitor is acertinib. The chemical structure and name of acertib are shown as: N-[(2S)-1-amino-3-(3,4-difluorophenyl)propan-2-yl]-5-chloro-4-( 4-Chloro-2-methylpyrazol-3-yl)furan-2-carboxamide

。

.

Triciribine

In one embodiment, the AKT inhibitor is triciribine. The chemical structure and name of Triciribine are shown as: (2R,3R,4S,5R)-2-(5-Amino-7-methyl-2,6,7,9,11-pentaazatricyclic [6.3.1.04,12] Dodeca-1(12),3,5,8,10-penten-2-yl)-5-(hydroxymethyl)oxolane-3,4-diol

。

.

SR13668

In one embodiment, the AKT inhibitor is SR13668. The chemical structure and name of SR13668 are shown as: Diethyl 6-methoxy-5,7-dihydroindolo[2,3-b]carbazole-2,10-dicarboxylate

。

.

A-443654

In one embodiment, the AKT inhibitor is A-443654. The chemical structure and name of A-443654 are shown as: (2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridine- 3-yl]oxypropan-2-amine

。

.

Deguelin

In one embodiment, the AKT inhibitor is deguelin. The chemical structure and name of deguelin are shown as: (1S,14S)-17,18-dimethoxy-7,7-dimethyl-2,8,21-trioxapentacyclo[12.8.0.0 ^{3 ,12} .0 ^4,9 .0 ^15,20 ]Docos-3(12),4(9),5,10,15,17,19-heptan-13-one

。

.

PHT-427

In one embodiment, the AKT inhibitor is PHT-427. The chemical structure and name of PHT-427 are shown as: 4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide

。

.

Miransertib (ARQ-092)

In one embodiment, the AKT inhibitor is milatitinib. The chemical structure and name of Miratinib are shown as: 3-[3-[4-(1-aminocyclobutyl)phenyl]-5-phenylimidazo[4,5-b]pyridine-2- Base]pyridin-2-amine

。

.

BAY1125976

-6-甲醯胺 In one embodiment, the AKT inhibitor is BAY1125976. The chemical structure and name of BAY1125976 are shown as: 2-[4-(1-aminocyclobutyl)phenyl]-3-phenylimidazo[1,2-b]pyridine

-6-formamide

。

.

TAS-117

In one embodiment, the AKT inhibitor is TAS-117. The chemical structure and name of TAS-117 are shown as: 3-amino-1-methyl-3-[4-(5-phenyl-8-oxa-3,6,12-triazatricyclo[7.4 .0.02,6]trideca-1(9),2,4,10,12-penten-4-yl)phenyl]cyclobutan-1-ol

。

.

MSC2363318A

In one embodiment, the AKT inhibitor is MSC2363318A. The chemical structure and name of MSC2363318A are shown as: 4-[[(1S)-2-(azetidin-1-yl)-1-[4-chloro-3-(trifluoromethyl)phenyl]B Base] amino] quinazoline-8-carboxamide

。

.

Triciribine Phosphate (VQD-002)

In one embodiment, the AKT inhibitor is triciribine phosphate. The chemical structure and name of tricitribine phosphate are shown as: dihydrogen phosphate [(2R,3S,4R,5R)-5-(5-amino-7-methyl-2,6,7,9,11- Pentaazatricyclo[6.3.1.04,12]dodeca-1(12),3,5,8,10-penten-2-yl)-3,4-dihydroxyoxolane-2- base] methyl ester

。

.

XL418

-1-基]-4-甲基-5-(2-吡咯啶-1-基乙胺基)苯基]-4,4,4-三氟丁-1-酮 In one embodiment, the AKT inhibitor is XL418. The chemical structure and name of XL418 are shown as: 1-[3-[4-(3-bromo-2H-pyrazolo[3,4-d]pyrimidin-4-yl)piper

-1-yl]-4-methyl-5-(2-pyrrolidin-1-ylethylamino)phenyl]-4,4,4-trifluorobutan-1-one

。

.

SC66

In one embodiment, the AKT inhibitor is SC66. The chemical structure and name of SC66 are shown as: (2E,6E)-2,6-bis(pyridin-4-ylmethylene)cyclohexan-1-one

。

.

Honokiol

In one embodiment, the AKT inhibitor is honokiol. The chemical structure and name of Honokiol are shown as: 2-(4-Hydroxy-3-prop-2-enylphenyl)-4-prop-2-enylphenyl

。

.

Vevorisertib (ARQ751)

In one embodiment, the AKT inhibitor is vivoltinib. The chemical structure and name of Vivotinib are shown as: N-[1-[3-[3-[4-(1-aminocyclobutyl)phenyl]-2-(2-aminopyridine-3- Base) imidazo[4,5-b]pyridin-5-yl]phenyl]piperidin-4-yl]-N-methylacetamide

。

.

PX-316

In one embodiment, the AKT inhibitor is PX-316. The chemical structure and name of PX-316 are shown as: [(2R)-2-methoxy-3-octadecyloxypropyl][(1R,2R,3S,4R,6R)-2,3,4, 6-Tetrahydroxycyclohexyl]hydrogen phosphate

。

.

API-1

In one embodiment, the AKT inhibitor is API-1. The chemical structure and name of API-1 are shown as: 4-amino 8-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxol-2-yl ]-5-oxopyrido[2,3-d]pyrimidine-6-carboxamide

。

.

ALM301

In one embodiment, the AKT inhibitor is ALM301. The chemical structure and name of ALM301 are shown as: 3-(3-(4-(1-aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridine-2- base) pyridin-2-amine

。

.

COTI-2

-1-硫代碳醯胺 In one embodiment, the AKT inhibitor is COTI-2. The chemical structure and name of COTI-2 are shown as: N-(6,7-dihydro-5H-quinolin-8-ylideneamino)-4-pyridin-2-ylpiper

-1-thiocarbamide

。

.

DC120

In one embodiment, the AKT inhibitor is DC120. The chemical structure and name of DC120 are shown as: N-[1-amino-3-(2,4-dichlorophenyl)propan-2-yl]-2-[2-(methylamino)pyrimidine-4- base]-1,3-thiazole-5-carboxamide

。

.

TD52

In one embodiment, the AKT inhibitor is TD52. The chemical structure and name of TD52 are shown as: 2-N,3-N-bis(3-ethynylphenyl)quinoxaline-2,3-diamine

。

.

Artemisinin

In one embodiment, the AKT inhibitor is artemisinin. The chemical structure and name of artemisinin are shown as: (1R,4S,5R,8S,9R,12S, 13R)-1,5,9-trimethyl-11,14,15,16-tetraoxatetracyclo [10.3.1.0 ^4,13 .0 ^8,13 ] Hexadecan-10-one

。

.

Guggulsterone

In one embodiment, the AKT inhibitor is bisabolone. The chemical structure and name of bisabolone are shown as: (8R,9S,10R,13S,14S, 17Z)-17-ethylidene-10,13-dimethyl-1,2,6,7,8, 9,11,12,14,15-Decahydrocyclopenta[a]phenanthrene-3,16-dione

。

.

Oridonin (NSC-250682)

In one embodiment, the AKT inhibitor is oridonin. The chemical structure and name of oridonin are shown as: (1S,2S,5S,8R, 9S,10S,11R,15S,18R)-9,10,15,18-tetrahydroxy-12,12-dimethyl Base-6-methylene-17-oxolane[7.6.2.1 ^5,8 .0 ^1,11 .0 ^2,8 ]octadec-7-one

。

.

Cenisertib (AS-703569)

-1-基)苯胺基]嘧啶-4-基]胺基]雙環[2.2.1]庚-5-烯-2-甲醯胺 In one embodiment, the AKT inhibitor is zeniseti. The chemical structure and name of zeniseti are shown as: (1S,2S,3R,4R)-3-[[5-fluoro-2-[3-methyl-4-(4-methylpiperene

-1-yl)anilino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide

。

.

3CAI

In one embodiment, the AKT inhibitor is 3CAI. The chemical structure and name of 3CAI are shown as: 2-Chloro-1-(1H-indol-3-yl)ethanone

。

.

Borussertib

In one embodiment, the AKT inhibitor is bolglutinib. The chemical structure and name of bolrutinib are shown as: N-[2-oxo-3-[1-[[4-(5-oxo-3-phenyl-6H-1,6-naphthyridine -2-yl)phenyl]methyl]piperidin-4-yl]-1H-benzimidazol-5-yl]prop-2-enamide

。

.

PF-AKT400

In one embodiment, the AKT inhibitor is PF-AKT400. The chemical structure and name of PF-AKT400 are shown as: N-[[(3S)-3-amino-1-(5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrole Pyridin-3-yl]methyl]-2,4-difluorobenzamide

。

.

Hu7691

In one embodiment, the AKT inhibitor is Hu7691. The chemical structure and name of Hu7691 are shown as: N-((3S,4S)-4-(3,4-difluorophenyl)piperidin-3-yl)-2-fluoro-4-(1-methyl- 1H-pyrazol-5-yl)benzoyl

。

.

Gossytin (Herbacetin)

烯-4-酮In one embodiment, the AKT inhibitor is gossytin. The chemical structure and name of cotton xanthin are shown as: 3,5,7,8-tetrahydroxy-2-(4-hydroxyphenyl)

en-4-one

。

.

Isoliquiritigenin

In one embodiment, the AKT inhibitor is isoliquiritigenin. The chemical structure and name of isoliquiritigenin are shown as: (E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one

。

.

Scutellarin

烯-7-基]氧基-3,4,5-三羥基

烷-2-甲酸In one embodiment, the AKT inhibitor is yellow acetin. The chemical structure and name of flavinin are shown as: (2S,3S,4S,5R,6S)-6-[5,6-dihydroxy-2-(4-hydroxyphenyl)-4-oxo

En-7-yl]oxy-3,4,5-trihydroxy

Alkane-2-carboxylic acid

。

.

Tehranolide

In one embodiment, the AKT inhibitor is Tenolid. The chemical structure and name of Tenolide are shown as: (1R,4R,5S,12S,13S)-4,13-dihydroxy-5,9-dimethyl-11,14,15-trioxatetracyclo [11.2.1.0 ^1,5 .0 ^8,12 ] Hexadecan-10-one

。

.

In some embodiments, the AKT inhibitor is selected from the group consisting of pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, Oridonin, gossinthin, tenoride, isoliquiritigenin, chrysanthemum, honokiol and pharmaceutically acceptable salts thereof. III. TIL Manufacturing Process -2A

An exemplary TIL process called Process 2A that incorporates some of these features is depicted in Figure 2, and some advantages of this embodiment of the invention over Process 1C are described in International Patent Publication WO2018/081473. One embodiment of Process 2A is shown in FIG. 1 .

As discussed herein, the invention may include steps associated with restimulation of cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplantation into a patient, and methods of testing such metabolic health. As generally outlined herein, TILs are generally obtained from a patient sample and manipulated to expand their number prior to transplantation into the patient. In some embodiments, TILs can optionally be genetically manipulated as discussed below.

In some embodiments, TILs can be stored cryopreserved. After thawing, it can also be restimulated to increase its metabolism prior to infusion into a patient.

In some embodiments, the first amplification (including the procedure called pre-REP and the procedure shown as step A in FIG. 1 ) is shortened to 3 to 14 days, and the second amplification (including the procedure called REP and that shown as Step B in Figure 1) was shortened to 7 to 14 days, as discussed in detail below and in the Examples and Figures. In some embodiments, the first amplification (such as the amplification described in step B in Figure 1) is shortened to 11 days, and the second amplification (such as the amplification described in Figure 1 step D) is shortened to 11 days. shortened to 11 days. In some embodiments, the combination of the first amplification and the second amplification (such as the amplifications described as steps B and D in FIG. 1 ) is shortened to 22 days, as detailed below and in the examples and figures. Discussed.

The following "step" designations A, B, C, etc. refer to FIG. 1 and to certain embodiments described herein. The order of the steps below and in Figure 1 is exemplary, and the present application and methods disclosed herein contemplate any combination or order of steps, as well as additional steps, repetitions of steps, and/or omission of steps. A. Step A : Obtaining Patient Tumor Samples

In general, TILs are initially obtained from patient tumor samples and subsequently expanded for further manipulation as described herein, optionally cryopreserved as outlined herein, restimulated, and optionally evaluated for expression as an indicator of TIL health. A larger group of phenotypes and metabolic parameters.

Patient tumor samples can be obtained using methods known in the art, typically by surgical resection, biopsy biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. In some embodiments, multifocal sampling is used. In some embodiments, surgical resection, needle biopsy, core needle biopsy, mini biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells includes multifocal sampling (i.e., from one or Multiple tumor sites and/or locations and samples obtained from one or more tumors at the same location or in close proximity). In general, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. A tumor sample can also be a liquid tumor, such as a tumor obtained from a hematological malignancy. A solid tumor can be lung tissue. In some embodiments, useful TILs are obtained from non-small cell lung cancer (NSCLC). Solid tumors may be skin tissue. In some embodiments, useful TILs are obtained from melanoma.

Once obtained, tumor samples are typically fragmented using sharps into pieces between 1 mm ³ and about 8 mm ³ , with about 2-3 mm ³ being particularly suitable. In some embodiments, TILs are grown from these fragments using enzymatic tumor digests. Such tumor lysates can be prepared in enzyme medium (such as Roswell Park Cancer Institute (Roswell Park Memorial Institute; RPMI) 1640 buffer, 2 mM glutamic acid, 10 mcg/mL gentamycin (gentamicine), 30 units/mL DNase and 1.0 mg/mL collagenase), followed by mechanical dissociation (eg, using a tissue dissociator). Tumor lysates can be generated by placing tumors in enzymatic medium and mechanically dissociating tumors for approximately 1 minute, followed by incubation at 37°C in 5% _CO for 30 minutes, followed by repeating mechanical dissociation and incubation under the aforementioned conditions Cycle until only small pieces of tissue remain. At the end of this process, if the cell suspension contains large numbers of red blood cells or dead cells, density gradient separation using FICOLL branched-chain hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in US Patent Application Publication No. 2012/0244133 Al, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in a method of expanding TILs or a method of treating cancer in any of the embodiments described herein.

The tumor dissociation enzyme mix may include one or more dissociation (digestion) enzymes such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, dispase (dispersed enzyme), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, protease XIV (protease) pronase), deoxyribonuclease I (DNase), trypsin inhibitors, any other dissociative or proteolytic enzymes, and any combination thereof.

In some embodiments, a resolvase is reconstituted from a lyophilized enzyme. In some embodiments, lyophilized enzymes are reconstituted in an amount of sterile buffer such as HBSS.

In some cases, collagenase (such as animal-derived-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the freeze-dried stock enzyme can be 2892 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, such as from about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL-about 350 PZ U/mL, about 100 PZ U/mL-about 300 PZ U/mL, about 150 PZ U/mL-about 400 PZ U/mL, about 100 PZ U/mL, about 150 PZ U/mL U/mL, about 200 PZ U/mL, about 210 PZ U/mL, about 220 PZ U/mL, about 230 PZ U/mL, about 240 PZ U/mL, about 250 PZ U/mL, about 260 PZ U /mL, about 270 PZ U/mL, about 280 PZ U/mL, about 289.2 PZ U/mL, about 300 PZ U/mL, about 350 PZ U/mL, or about 400 PZ U/mL.

In some embodiments, dispase is reconstituted in 1 ml sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after reconstitution, the neutral protease stock solution ranges from about 100 DMC/mL to about 400 DMC/mL, such as from about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL - about 350 DMC/mL, about 100 DMC/mL - about 300 DMC/mL, about 150 DMC/mL - about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC /mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 ml sterile HBSS or another buffer. The concentration of lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, such as about 1 KU/mL, about 2 KU/mL, about 3 KU/mL mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, stock solutions of enzymes are variable and concentrations may need to be determined. In some embodiments, the concentration of the lyophilized stock solution can be verified. In some embodiments, the final amount of enzyme added to the digest mixture is adjusted based on the determined concentration of the stock solution.

In some embodiments, the enzyme mix includes about 10.2 μl of dispase (0.36 DMC U/mL), 21.3 μl of collagenase (1.2 PZ/mL), and 250 μl of deoxyribonuclease I in about 4.7 mL of sterile HBSS (200 U/mL).

As noted above, in some embodiments, TILs are derived from solid tumors. In some embodiments, solid tumors are not fragmented. In some embodiments, solid tumors are not fragmented and whole tumors are enzymatically lysed. In some embodiments, tumors are lysed in an enzyme mixture comprising collagenase, DNase, and hyaluronidase. In some embodiments, tumors are lysed in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1 to 2 hours. In some embodiments, tumors are lysed in an enzyme mixture comprising collagenase, DNase, and hyaluronidase at 37° C., 5% CO ₂ for 1 to 2 hours. In some embodiments, tumors are lysed in an enzyme mixture comprising collagenase, DNase, and hyaluronidase at 37° C., 5% CO ₂ , with rotation, for 1 to 2 hours. In some embodiments, tumor lines were lysed overnight with constant rotation. In some embodiments, tumors are lysed overnight at 37°C, 5% _CO2 , with constant rotation. In some embodiments, whole tumors are combined with enzymes to form a tumor digestion reaction mixture.

In some embodiments, tumors are reconstituted with lyophilized enzymes in sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of collagenase is a 100 mg/mL 10× working stock solution.

In some embodiments, the enzyme mixture comprises DNase. In some embodiments, the working stock solution of DNase is 10,000 IU/ml 10× working stock solution.

In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is a 10 mg/mL 10× working stock solution.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase, 1000 IU/mL DNase, and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase, 500 IU/mL DNA, and 1 mg/mL hyaluronidase.

Generally, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

In some embodiments, fragmentation includes physical fragmentation, including, for example, segmentation and dissolution. In some embodiments, segments are physical segments. In some embodiments, a segment is a segmentation. In some embodiments, the fragments are by dissolution. In some embodiments, TILs can be cultured initially from enzymatic tumor digests and tumor fragments obtained by digesting or fragmenting tumor samples obtained from patients.

In some embodiments, when the tumor is a solid tumor, the tumor undergoes physical fragmentation after obtaining a tumor sample, eg, in Step A (as provided in Figure 1 or Figure 8). In some embodiments, fragmentation occurs prior to cryopreservation. In some embodiments, fragmentation occurs following cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each vessel for the first expansion. In some embodiments, tumors are fragmented and 30 or 40 fragments or pieces are placed in each vessel for the first expansion. In some embodiments, tumors are fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the plurality of fragments comprises from about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm ³ . In some embodiments, the plurality of segments comprises about 30 to about 60 segments having a total volume of about 1300 mm ³ to about 1500 mm ³ . In some embodiments, the plurality of segments comprises about 50 segments having a total volume of about 1350 mm ³ . In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

In some embodiments, TILs are obtained from tumor fragments. In some embodiments, tumor fragments are obtained by sharps segmentation. In some embodiments, the tumor fragment is between about 1 mm ³ and 10 mm ³ . In some embodiments, the tumor fragment is between about 1 mm ³ and 8 mm ³ . In some embodiments, the tumor fragment is about 1 mm ³ . In some embodiments, the tumor fragment is about 2 mm ³ . In some embodiments, the tumor fragment is about 3 mm ³ . In some embodiments, the tumor fragment is about 4 mm ³ . In some embodiments, the tumor fragment is about 5 mm ³ . In some embodiments, the tumor fragment is about 6 mm ³ . In some embodiments, the tumor fragment is about 7 mm ³ . In some embodiments, the tumor fragment is about 8 mm ³ . In some embodiments, the tumor fragment is about 9 mm ³ . In some embodiments, the tumor fragment is about 10 mm ³ . In some embodiments, the tumor is 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumor is 1 mm x 1 mm x 1 mm. In some embodiments, the tumor is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor is 4 mm x 4 mm x 4 mm.

In some embodiments, tumors are resected so as to minimize the amount of hemorrhage, necrosis, and/or fatty tissue in each patch. In some embodiments, tumors are resected to minimize the amount of bleeding tissue on each patch. In some embodiments, tumors are resected to minimize the amount of necrotic tissue in each patch. In some embodiments, tumors are resected to minimize the amount of adipose tissue on each patch.

In some embodiments, tumor fragmentation is performed so as to maintain tumor internal structure. In some embodiments, tumor fragmentation is performed without the sawing action of a scalpel. In some embodiments, TILs are obtained from tumor lysates. In some embodiments, by incubating in an enzyme medium (such as but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamycin, 30 U/mL DNase, and 1.0 mg/mL collagenase), followed by Mechanical dissociation (GentleMACS, Miltenyi Biotechnology, Auburn, CA) was performed to generate tumor lysates. Tumors can be dissociated mechanically for about 1 minute after placing them in the enzymatic medium. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically disrupted again for about 1 minute. Tumors were mechanically disrupted a third time for approximately 1 min after an additional 30 min incubation at 37°C in 5% _CO2 . In some embodiments, if large pieces of tissue remain after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to _the sample, with or without further incubation at 37°C in 5% CO for 30 minutes. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation can be performed using Ficoll to remove these cells.

In some embodiments, the cell suspension collected prior to the first expansion step is referred to as a "primary cell population" or a "freshly collected" cell population.

In some embodiments, cells are optionally frozen after sample collection and stored frozen prior to proceeding to expansion as described in Step B, which is described in further detail below and illustrated in FIGS. 1 and 8 . 1. T cells and TIL in pleural effusion

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cells or TILs for expansion according to the procedures described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of T cells or TILs for expansion according to the procedures described herein is a pleural effusion-derived sample. See, eg, the methods described in US Patent Publication US 2014/0295426, which is hereby incorporated by reference in its entirety for all purposes.

In some embodiments, any pleural fluid or pleural effusion suspected of and/or containing TIL may be used. Such samples may be derived from primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, the sample may be derived from a secondary metastatic cancer cell originating from another organ (eg, breast, ovary, colon, or prostate). In some embodiments, the sample used in the amplification methods described herein is pleural exudate. In some embodiments, the sample used in the amplification methods described herein is a pleural transudate. Other biological samples may include other serum fluids containing TILs, including, for example, ascitic fluid or pancreatic cyst fluid from the abdomen. Ascites and pleural fluids involve very similar chemical systems; both the abdomen and the lungs have mesothelial cell lines and fluid forms in the thoracic and peritoneal cavities in the same malignancy event, and in some embodiments, such fluids contain TIL. In some embodiments of the present invention exemplifying pleural fluid, the same method can be performed using ascites or other cystic fluid containing TIL to obtain similar results.

In some embodiments, pleural fluid is removed directly from the patient in an unprocessed form. In some embodiments, unprocessed pleural fluid is placed in standard blood collection tubes, such as EDTA or heparin tubes, prior to further processing steps. In some embodiments, unprocessed pleural fluid was placed in standard CellSave® tubes (Veridex) prior to further processing steps. In some embodiments, samples are placed in CellSave tubes immediately after collection from the patient to avoid a reduction in the number of viable TILs. If retained in untreated pleural fluid, the number of viable TILs may decrease significantly within 24 hours, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

In some embodiments, samples of pleural fluid from selected individuals can be diluted. In some embodiments, the dilution is 1:10 pleural fluid to diluent. In other embodiments, the dilution is 1:9 pleural fluid to diluent. In other embodiments, the dilution is 1:8 pleural fluid to diluent. In other embodiments, the dilution is 1:5 pleural fluid to diluent. In other embodiments, the dilution is 1:2 pleural fluid to diluent. In other embodiments, the dilution is 1:1 pleural fluid to diluent. In some embodiments, the diluent includes saline, phosphate buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, samples were placed in CellSave tubes immediately after collection and dilution from the patient to avoid reduction of viable TILs, which may be present at 4°C if retained in untreated pleural fluid. Visible reduction within 24 to 48 hours. In some embodiments, the pleural fluid sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal from the patient and dilution. In some embodiments, the pleural fluid sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal from the patient and dilution at 4°C.

In other embodiments, the pleural fluid sample is concentrated by conventional means prior to further processing steps. In some embodiments, preconditioning of pleural fluid is preferred in cases where it must be stored frozen for transport to the laboratory performing the method or for subsequent analysis (e.g., after 24 to 48 hours after collection) . In some embodiments, the pleural fluid sample is prepared by centrifuging the pleural fluid sample after it is removed from the individual and resuspending the centrifugate or pellet in buffer. In some embodiments, pleural fluid samples are centrifuged and resuspended multiple times before being frozen for shipping or later analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated by using filtration methods prior to further processing steps. In some embodiments, samples of pleural fluid used in further processing are prepared by filtering the fluid through a filter having a known and substantially uniform pore size that allows pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the pores in the membrane may be at least 4 μM in diameter. In other embodiments, the pore size can be 5 μM or larger, and in other embodiments, can be any of 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. Following filtration, cells retained by the membrane, including TIL, can be washed from the membrane into a suitable physiologically acceptable buffer. Cells (including TILs) concentrated in this way can then be used in the contacting step of the method.

In some embodiments, a pleural fluid sample (including, for example, unprocessed pleural fluid), diluted pleural fluid, or a resuspended cell pellet is contacted with a lysing agent that differentially lyses cells present in the sample. Anucleated red blood cells. In some embodiments, this step is performed prior to further processing steps where the pleural fluid contains a large number of RBCs. Suitable lysis reagents include a single lysis reagent or a lysis reagent and a quenching reagent, or a lysis reagent, a quencher and a fixation reagent. Suitable dissolution systems are commercially available and include the BD Pharm Lyse™ system (Becton Dickenson). Other dissolution systems include the Versalyse™ system, the FACSlyse™ system (BD Medical), the Immunoprep™ system, or the Erythrolyse II system (Beckman Coulter, Inc.) or the ammonium chloride system. In some embodiments, the lysing agent may vary with the prevailing requirements, which are efficient lysis of erythrocytes and conservation of TILs and phenotypic properties of TILs in pleural fluid. In addition to employing a single reagent for lysis, a lysis system suitable for use in the methods described herein may include a second reagent, such as a second reagent that quenches or retards the effect of the lysis reagent during the remaining steps of the method, such as Stabilyse™ Reagents (Beckman Coulter). Depending on the choice of solubilizing reagent or the preferred practice of the method, conventional immobilizing reagents may also be used.

In some embodiments, untreated, diluted, or multiple centrifuged or processed pleural fluid samples as described above are cryopreserved at a temperature of about -140° C., followed by further processing and/or amplification as provided herein . 2. Pre-selection of CD39/CD69 double negative and CD39/CD69 double KO

According to some methods of the invention, TILs are preselected prior to the first amplification for (i) CD39/CD69 double negative, (ii) CD39/CD69 double knockout, or (iii) (i) and (ii) combination. In some embodiments, there is also an optional pre-selection step for PD-1.

In some embodiments, a minimum of 3,000 TILs need to be seeded to the first expansion. In some embodiments, the pre-selection step yields a minimum of 3,000 TILs. In some embodiments, a minimum of 4,000 TILs need to be seeded for the first expansion. In some embodiments, the pre-selection step yields a minimum of 4,000 TILs. In some embodiments, a minimum of 5,000 TILs need to be seeded for the first expansion. In some embodiments, the pre-selection step yields a minimum of 5,000 TILs. In some embodiments, a minimum of 6,000 TILs need to be seeded to the first expansion. In some embodiments, the pre-selection step yields a minimum of 6,000 TILs. In some embodiments, a minimum of 7,000 TILs need to be seeded to the first expansion. In some embodiments, the pre-selection step yields a minimum of 7,000 TILs. In some embodiments, a minimum of 8,000 TILs need to be seeded for the first expansion. In some embodiments, the pre-selection step yields a minimum of 8,000 TILs. In some embodiments, a minimum of 9,000 TILs need to be seeded to the first expansion. In some embodiments, the pre-selection step yields a minimum of 9,000 TILs. In some embodiments, a minimum of 10,000 TILs need to be seeded for the first expansion. In some embodiments, the pre-selection step yields a minimum of 10,000 TILs. In some embodiments, a minimum of 20,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 20,000 TILs. In some embodiments, a minimum of 30,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 30,000 TILs. In some embodiments, a minimum of 40,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 40,000 TILs. In some embodiments, a minimum of 50,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 50,000 TILs. In some embodiments, a minimum of 60,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 60,000 TILs. In some embodiments, a minimum of 70,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 70,000 TILs. In some embodiments, a minimum of 80,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 80,000 TILs. In some embodiments, a minimum of 90,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 90,000 TILs. In some embodiments, a minimum of 100,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 100,000 TILs. In some embodiments, the cells are grown or expanded to a density of 200,000. In some embodiments, the cells are grown or expanded to a density of 200,000 to provide about 2e8 TILs to initiate the second expansion. In some embodiments, the cells are grown or expanded to a density of 150,000. In some embodiments, the cells are grown or expanded to a density of 150,000 to provide about 2e8 TILs to initiate the second expansion. In some embodiments, the cells are grown or expanded to a density of 250,000. In some embodiments, the cells are grown or expanded to a density of 250,000 to provide about 2e8 TILs to initiate the second expansion. In some embodiments, the minimum cell density is 10,000 cells to give 10e6 to initiate the second expansion. In some embodiments, the seeding density of 10e6 used to initiate the second expansion can result in greater than 1e9 TILs.

In some embodiments, the TILs used for the first amplification are (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double gene knockout, or (iii) (i) and A combination of (ii) (eg, after preselection and before first amplification). In some embodiments, the TILs used for the first amplification are at least 75% (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) ( Combination of i) and (ii); at least 80% (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) (i) and (ii) combination; at least 85% (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) a combination of (i) and (ii); at least 90% (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) a combination of (i) and (ii); at least 95% of (i) CD39/CD69 Double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) a combination of (i) and (ii); at least 98% of (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double gene knockout, or (iii) a combination of (i) and (ii); or at least 99% (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) a combination of (i) and (ii) (eg, after pre-selection and before first amplification).

In some embodiments, pre-diagnosis of CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO is performed by staining primary cell populations, whole tumor digests, and/or whole tumor cell suspension TILs with anti-CD39 and anti-CD69 antibodies. choose. In some embodiments, the anti-CD39 and anti-CD69 antibodies are polyclonal antibodies, such as mouse anti-human CD39 and CD69 polyclonal antibodies, goat anti-human CD39 and CD69 polyclonal antibodies, and the like. In some embodiments, the anti-CD39 and anti-CD69 antibodies are monoclonal antibodies.

In some embodiments, the anti-CD39 antibody used in the preselection is associated with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells expressing CD39 combined. In some embodiments, the anti-CD69 antibody used in the preselection is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% cell binding.

In some embodiments, the patient has been treated with an anti-PD-1 antibody. In some embodiments, the individual has not received anti-PD-1 antibody treatment. In some embodiments, the individual has not been treated with an anti-PD-1 antibody. In some embodiments, the individual has been previously treated with a chemotherapeutic agent. In some embodiments, the individual has previously been treated with a chemotherapeutic agent, but is no longer being treated with that chemotherapeutic agent. In some embodiments, the individual has been treated with a chemotherapeutic agent, or has been treated with an anti-PD-1 antibody. In some embodiments, the individual has been treated with a chemotherapeutic agent and has been treated with an anti-PD-1 antibody. In some embodiments, the patient has not received anti-PD-1 antibody therapy. In some embodiments, the individual is cancer-naïve or treated with a chemotherapeutic agent but not treated with an anti-PD-1 antibody. In some embodiments, the individual is treatment naïve and chemotherapeutic agent treated but not anti-PD-1 antibody treated.

In some embodiments, cell sorting methods are used for preselection. In some embodiments, the cell sorting method is a flow cytometric method, such as flow activated cell sorting (FACS). In some embodiments, negative circles are based on FMO. In some embodiments, FACS banking is set after the step of obtaining and/or receiving a first TIL population from a resected tumor of an individual by processing a tumor sample obtained from the individual into tumor fragments. In some embodiments, circles are set for each sort. In some embodiments, a circle is configured for each PBMC sample. In some embodiments, a circle is configured for each PBMC sample. In some embodiments, the circled template is set from PBMCs every 10 days, 20 days, 30 days, 40 days, 50 days, or 60 days. In some embodiments, a circled template is set every 60 days from PBMCs. In some embodiments, the circled template is set for each PBMC sample every 10 days, 20 days, 30 days, 40 days, 50 days, or 60 days. In some embodiments, a circled template is set for each PBMC sample every 60 days.

In some embodiments, the pre-selection involves selecting from the first TIL population (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double gene knockout, or (iii) (i) and The combination of (ii) to obtain a TIL population comprising a selected TIL population from the first TIL population that is at least 11.27% to 74.4% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL. In some embodiments, the first TIL population is at least 10% to 80% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs, at least 20% to 80% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 20% to 80% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 30% to 80% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 40% to 80 % CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 50% to 80% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 10% to 70% CD39/CD69 double negative and/ or CD39 ^LO /CD69 ^LO TIL, at least 20% to 70% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 30% to 70% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL or At least 40% to 70% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL.

In some embodiments, the selecting step (e.g., pre-selecting and/or selecting for CD39/CD69 double negative cells) comprises the step of: (i) exposing the first TIL population and the PBMC population to an excess of a single CD39- and CD69-binding Strain anti-CD39 IgG and anti-CD69 IgG antibodies, (ii) add an excess of anti-IgG antibody bound to the fluorophore, (iii) based on the intensity of the fluorophore detected in the PBMC population, in the first CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL populations in TIL populations were obtained by intensities of fluorophores detected in CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs, e.g. by fluorescent Photoactivated cell sorting (FACS) was performed.

In some embodiments, at least 70% of the population of CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs. In some embodiments, at least 80% of the population of TILs enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs. In some embodiments, at least 90% of the population of TILs enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO are PD-1 positive TILs. In some embodiments, at least 95% of the population of TILs enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs. In some embodiments, at least 99% of the population of TILs enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs. In some embodiments, 100% of the population of TILs enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs.

In some embodiments, the selection step as exemplified in step A2 of FIG. 8 comprises the steps of: (i) exposing the first TIL population to excess monoclonal anti-CD39 and anti-CD69 IgG antibodies, (ii) adding excess and Fluorophore-conjugated anti-IgG antibody, and (iii) fluorophore-based flow cytometric sorting for (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double Gene knockout, or (iii) enrichment for a combination of the TIL populations of (i) and (ii).

In some embodiments, the circle method of Example 15 is used. To determine whether TILs derived from a tumor sample are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , one skilled in the art can use TILs corresponding to peripheral T cells obtained from blood samples obtained from one or more healthy human individuals. The reference value of CD39 and/or CD69 expression. CD39/CD69 positive cells in a reference sample can be defined using a fluorescence minus one control and a matched isotype control. In some embodiments, immunostaining for CD39/CD69 in tumor-derived TILs is established using the expression of CD39/CD69 measured in CD3+/CD39+/CD69+ peripheral T cells (e.g., reference cells) from healthy individuals Threshold or cut-off value for intensity. The cut-off value can be defined as the maximum intensity of CD39/CD69 immunostaining for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL. Therefore, TILs with CD39/CD69 expression equal to or below the cut-off value can be considered as CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO cells. In some instances, CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs represent TILs with the lowest intensity of CD39/CD69 immunostaining corresponding to up to 1% or less of total CD3+ cells. In other cases, CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs represent TILs with the lowest intensity of CD39/CD69 immunostaining corresponding to a maximum of 0.75% or less of total CD3+ cells. In some instances, CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs represent TILs with the lowest intensity of CD39/CD69 immunostaining corresponding to a maximum of 0.50% or less of total CD3+ cells. In one example, CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs represent TILs with the lowest intensity of CD39/CD69 immunostaining corresponding to a maximum of 0.25% or less of total CD3+ cells.

In some embodiments, the protein kinase B (AKT) inhibitor (AKTi) method of Example 16 is used. In some embodiments, the TIL population is cultured in a medium containing an AKT inhibitor to obtain a CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population. In some embodiments, the AKT inhibitor is selected from the group consisting of pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, Oridonin, gossinthin, tenoride, isoliquiritigenin, chrysanthemum, honokiol and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is pataxerti. In some embodiments, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, About 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM or about 5 μM in the culture medium of pataxerti Cultivate TIL populations. a. Fluorophores

In some embodiments, primary cell population TILs are stained with a mixture comprising anti-CD39 and anti-CD69 antibodies linked to a fluorophore and anti-CD3 antibody linked to a fluorophore. In some embodiments, primary cell population TILs are stained with a cocktail comprising anti-CD39 and anti-CD69 antibodies linked to fluorophores (eg, PE, live/dead purple) and anti-CD3-FITC. In some embodiments, primary cell population TILs are stained with a mixture comprising anti-CD39 conjugated to a first fluorophore and anti-CD69 antibody conjugated to a second fluorophore, anti-CD3 conjugated to a third fluorophore Antibodies and live/dead blue dye (such as available from ThermoFisher, MA, Cat. No. L23105), where the primary, secondary, and tertiary antibodies are distinct and capable of individual detection. In some embodiments, following incubation with anti-CD39 and anti-CD69 antibodies, (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) Combinations of (i) and (ii) cells for expansion according to the first amplification described herein (eg, in Figure 8E and/or Figure 8F and/or Figure 8G).

In some embodiments, primary cell population TILs are stained with a mixture comprising anti-CD39 and anti-CD69 antibodies linked to a fluorophore and anti-CD3 antibody linked to a fluorophore. In some embodiments, primary cell population TILs are stained with a cocktail comprising anti-CD39 and anti-CD69 antibodies linked to a fluorophore (eg, PE, live/dead purple) and anti-CD3-PE-Cy7. In some embodiments, primary cell population TILs are stained with a mixture comprising anti-CD39-FITC and anti-CD69-PE, anti-CD3-PE-Cy7 and live/dead blue dye (ThermoFisher, MA, cat# L23105). In some embodiments, following incubation with anti-CD39 and anti-CD69 antibodies, (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) Combinations of (i) and (ii) cells for use in priming as described herein (e.g., in step B of process CD39/CD69 GEN 3 in Figure 8E and/or Figure 8F and/or Figure 8G ) 1. Amplification of expansion.

In some embodiments, fluorophores include, but are not limited to, PE (Phycoerythrin), APC (Allophycocyanin), PerCP (Dinoxanthin Chlorophyll Protein), DyLight 405, Alexa Fluor 405, Pacific Blue, Alexa Fluor 488, FITC (fluorescent isothiocyanate), DyLight 550, Alexa Fluor 647, DyLight 650 and Alexa Fluor 700. In some embodiments, fluorophores include, but are not limited to, PE-Alexa Fluor® 647, PE-Cy5, PerCP-Cy5.5, PE-Cy5.5, PE-Alexa Fluor® 750, PE-Cy7, and APC -Cy7. In some embodiments, fluorophores include, but are not limited to, luciferin dyes. Examples of luciferin dyes include, but are not limited to, 5-carboxyluciferin, luciferin-5-isothiocyanate, and 6-carboxyluciferin, 5,6-dicarboxyluciferin, 5- (and 6)-sulfoluciferin, pluciferin, succinyl luciferin, 5-(and 6)-carboxy SNARF-1, carboxyluciferin sulfonate, carboxyluciferin zwitterion , Carboxyluciferin quaternary ammonium, carboxyluciferin phosphonate, carboxyluciferin GABA, 5'(6')-carboxyluciferin, carboxyluciferin-cys-Cy5 and luciferin glutathione peptide. In some embodiments, the fluorescent moiety is rose bengal dye. Examples of rose bengal dyes include, but are not limited to, tetramethyl rose bengal-6-isothiocyanate, 5-carboxytetramethyl rose bengal, 5-carboxyrhodol derivatives, carboxy rose bengal 110, Tetramethyl and Tetraethyl Rose Bengal, Diphenyldimethyl and Diphenyldiethyl Rose Bengal, Dinaphthyl Rose Bengal, Rose Bengal 101 Sulfonyl Chloride (sold under the trade name TEXAS RED®). In some embodiments, the fluorescent moiety is a cyanine dye. Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7. B. Step B : First Amplification

In some embodiments, the methods of the invention provide for obtaining young TILs that are capable of providing increased replication cycles upon administration to an individual/patient and thus may provide superior Additional healing benefit of more rounds of replicated TIL). The characteristics of young TILs have been described in the literature, for example in Donia et al., Scand. J. Immunol. 2012, 75, 157-167; Dudley et al., Clin Cancer Research ( Clin. Cancer Res. )” 2010, 16, 6122-6131; Huang et al., “Journal of Immunotherapy ( J. Immunother. )” 2005, 28 , 258-267; Besser et al., “Clinical Cancer Research” 2013 , 19 , OF1-OF9; Besser et al., Journal of Immunotherapy 2009 , 32 :415-423; Robbins et al., Journal of Immunology 2004 , 173, 7125-7130; Shen et al., Journal of Immunotherapy , 2007, 30, 123-129; Zhou et al., Journal of Immunotherapy 2005, 28, 53-62; and Tran et al., Journal of Immunotherapy, 2008 , 31 , 742-751, each cited by way incorporated into this article.

The diverse antigen receptors of T and B lymphocytes are generated by somatic recombination of a limited but large number of gene segments. These gene segments: V (variable region), D (diversity region), J (junction region) and C (constant region) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCR). The present invention provides a method for generating TILs that exhibit and increase the diversity of the T cell repertoire. In some embodiments, TILs obtained by the methods of the invention exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained by the methods of the invention exhibit increased T cell repertoire diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein. Other methods include, for example, methods other than those implemented in FIG. 1 or FIG. 8 . In some embodiments, TILs obtained by the methods of the invention exhibit a T cell repertoire compared to freshly collected TILs and/or TILs prepared using a method referred to as Method 1C as exemplified in Figure 5 and/or Figure 6 Diversity increases. In some embodiments, the TILs obtained in the first expansion exhibit increased T cell repertoire diversity. In some embodiments, increasing diversity is increasing immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, present in immunoglobulin heavy chains. In some embodiments, the diversity is present in immunoglobulins, in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and/or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, TCRab (ie, TCRα/β) expression is increased.

After dissection or digestion of tumor fragments, eg as described in Figure 1 or Step A of Figure 8, the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs relative to tumor and other cells. In some embodiments, tumor digests were grown in medium containing 6000 IU/mL IL-2 inactivated human AB serum in 2 mL wells. This primary cell population is cultured for a period of several days, typically 3 to 14 days, to produce a bulk TIL population, typically about 1 x ¹⁰⁸ host TIL cells. In some embodiments, this primary cell population is cultured for a period of 7 to 14 days to produce a bulk TIL population, typically about 1 x ¹⁰⁸ host TIL cells. In some embodiments, this primary cell population is cultured for a period of 10 to 14 days to produce a bulk TIL population, typically about 1 x ¹⁰⁸ host TIL cells. In some embodiments, this primary cell population is cultured for a period of about 11 days to produce a bulk TIL population, typically about 1 x ¹⁰⁸ host TIL cells.

In some embodiments, expansion of TILs may use an initial bulk TIL expansion step as described below and herein (such as those described in Figure 1 or Step B of Figure 8, which may include what is known as a pre-REP process), followed by step D below and a second amplification as described herein (step D, including a process known as the Rapid Expansion Protocol (REP) step), followed by optional cryopreservation, and then A second step D (including a process known as the restimulation REP step) is performed as described below and herein. TILs obtained from this process can optionally be characterized for phenotypic characteristics and metabolic parameters as described herein.

In the example where TIL cultures are initiated in 24-well dishes, for example, using Costar 24-well cell culture clusters, flat bottom (Corning Incorporated, Corning, NY, each well can be seeded with 1 x ¹⁰ tumor digest cells or containing 2 mL of complete medium (CM) with IL-2 (6000 IU/mL; Chiron Corp., Emeryville, CA) for one tumor fragment. In some embodiments, the tumor fragment is ^between about ¹ mm and 10 mm .

In some embodiments, the first expansion medium is referred to as "CM" (short for medium). In some embodiments, the CM of Step B consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL Gentamycin. In the embodiment of initiating culture in a gas permeable bottle (for example, G-REX10; Wilson Wolf Manufacturing, New Brighton, MN) with a capacity of 40 mL and a gas permeable silicon bottom of 10 cm ² , each bottle was loaded with 10-40× 10-40 mL CM with IL-2 for ¹⁰⁶ live tumor digest cells or 5-30 tumor fragments. Both G-REXREX10 and 24-well plates were cultured in a humidified incubator at 37°C, 5% CO ₂ and 5 days after the initiation of culture, half of the medium was removed and replaced with fresh CM and IL-2, and the After day 5, half of the medium was changed every 2-3 days.

In some embodiments, the medium used in the expansion processes disclosed herein is a serum-free medium or a defined medium. In some embodiments, serum-free or defined media comprise basal cell culture media and serum supplements and/or serum substitutes. In some embodiments, serum-free or defined media are used to prevent and/or reduce experimental variation in part due to batch-to-batch variation of serum-containing media.

In some embodiments, serum-free or defined media comprise basal cell culture media and serum supplements and/or serum substitutes. In some embodiments, basal cell culture media include, but are not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T cell expansion Xeno-free medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI Growth Medium, and Ischoff's Modified Dulbecco's Medium.

In some embodiments, serum supplements or serum replacements include, but are not limited to, one or more of: CTS™ OpTmizer T Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagens Protein precursors, one or more antibiotics and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L - Phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine , reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and trace elements containing Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ , Ni ²⁺ , Rb ⁺ , Sn ²⁺ and Zr ⁴⁺ compounds. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some embodiments, CTS™ OpTmizer™ T Cell Immune Cell Serum Replacement is used with conventional growth media including, but not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dahl Burke's medium.

In some embodiments, the total serum replacement concentration (vol %) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, based on the total volume of the serum-free or defined medium. 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In some embodiments, the total serum replacement concentration is about 3% of the total volume of serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of serum-free or defined medium.

In some embodiments, the serum-free or defined medium is a CTS™ OpTmizer™ T cell expansion SFM (Thermo Fisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 µM.

In some embodiments, the defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (Thermo Fisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L - Glutamine. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 6000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 3000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 µM.

In some embodiments, serum-free medium or synthetic medium is supplemented with a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM or 4 mM to about 5 mM glutamine (ie, GlutaMAX®). In some embodiments, the serum-free medium or synthetic medium is supplemented with glutamine (ie, GlutaMAX®) at a concentration of about 2 mM.

In some embodiments, serum-free medium or defined medium is supplemented with a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM , 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or About 65 mM of 2-mercaptoethanol. In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the medium is 55 µM.

In some embodiments, defined media as described in International PCT Publication No. WO/1998/030679, incorporated herein by reference, find use in the present invention. In this publication, a serum-free eukaryotic cell culture medium is described. Serum-free eukaryotic cell culture media include basal cell culture media supplemented with serum-free supplements capable of supporting the growth of cells in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises one or more components selected from the group consisting of, or is obtained by combining one or more components selected from the group consisting of: one or more albumins or albumin substitutes one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, One or more trace elements and one or more antibiotics. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin Ferritin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L - Phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine , reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and trace elements containing Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ , Ni ²⁺ , Rb ⁺ , Sn ²⁺ and Zr ⁴⁺ compounds. In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640 , F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dulbecco's Medium.

In some embodiments, it is determined that the concentration of glycine in the medium is in the range of about 5 to 200 mg/L, the concentration of L-histidine is in the range of about 5 to 250 mg/L, and the concentration of L-isoleucine is The concentration of L-methionine is about 5 to 300 mg/L, the concentration of L-methionine is about 5 to 200 mg/L, the concentration of L-phenylalanine is about 5 to 400 mg/L, and the concentration of L-proline is about 1 to 1000 mg/L, the concentration of L-hydroxyproline is about 1 to 45 mg/L, the concentration of L-serine is about 1 to 250 mg/L, and the concentration of L-threonine is about 10 to 500 mg/L, the concentration of L-tryptophan is about 2 to 110 mg/L, the concentration of L-tyrosine is about 3 to 175 mg/L, and the concentration of L-valine is about 5 to 500 mg/L, the concentration of thiamine is about 1 to 20 mg/L, the concentration of reduced glutathione is about 1 to 20 mg/L, and the concentration of L-ascorbic acid-2-phosphate is about 1 to 200 mg /L, the concentration of iron-saturated transferrin is about 1 to 50 mg/L, the concentration of insulin is about 1 to 100 mg/L, the concentration of sodium selenite is about 0.000001 to 0.0001 mg/L, and albumin ( For example, the concentration of AlbuMAX® I) is about 5000 to 50,000 mg/L.

In some embodiments, the non-trace element fraction of the synthetic medium is present in the concentration ranges listed in the column headed "Concentration Range in 1X Medium" in Table 4 below. In other embodiments, the non-trace element components of the defined medium are present at the final concentrations listed in Table 4 in the column headed "Preferred Embodiments of 1X Medium". In other embodiments, the defined medium is a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trivial fraction ingredients of the type and concentration listed in the column titled "Preferred Embodiments of Supplements" in Table 4 below.

In some embodiments, the osmolarity of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmolarity is between about 280 and 310 mOsmol. In some embodiments, defined medium is supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. Defined media can be further supplemented with L-glutamine (at a final concentration of approximately 2 mM), one or more antibiotics, non-essential amino acids (NEAA; at a final concentration of approximately 100 μM), 2-mercaptoethanol (at a final concentration of about 100 μM).

In some embodiments, the defined medium described in Smith et al., " Clin Transl Immunology ", 4(1) 2015 (doi: 10.1038/cti.2014.31) can be used in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as basal cell culture medium supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

In some embodiments, the cell culture medium in the first and/or second gas permeable container is unfiltered. The use of unfiltered cell culture media simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second gas-permeable container lacks β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

Following preparation of tumor fragments, the resulting cells (ie, fragments) are cultured in serum containing IL-2 under conditions that favor the growth of TILs relative to tumor and other cells. In some embodiments, tumor digests were prepared in 2 mL wells in the presence of inactivated human AB serum (or in some cases, as outlined herein, in the presence of a population of APC cells) and 6000 IU/mL IL -2 culture medium. This primary cell population is cultured for a period of several days, typically 10 to 14 days, to produce a bulk TIL population, typically about 1 x ¹⁰⁸ host TIL cells. In some embodiments, the growth medium comprises IL-2 or a variant thereof during the first expansion. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20 to 30 x ¹⁰⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20 x ¹⁰⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 25 x ¹⁰⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 30 x ¹⁰⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4 to 8×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5 to 7×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6×10 ⁶ IU/mg IL-2. In some embodiments, IL-2 stock solutions are prepared as described in Example 5. In some embodiments, the first expansion medium comprises about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6000 IU/mL IL-2 or about 5,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL , about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL- 2. In some embodiments, the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, at Between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or 8000 IU/mL of IL-2.

In some embodiments, the first expansion medium comprises about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 200 IU/mL IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15.

In some embodiments, the first expansion medium comprises about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21 twenty one. In some embodiments, the first expansion medium comprises about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 2 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21.

In some embodiments, the cell culture medium comprises an anti-CD3 agonist antibody, such as an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL , about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 µg/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium does not contain an OKT-3 antibody. In some embodiments, the OKT-3 antibody is murozumab. See eg Table 1.

In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: Urelumab, Utumumab, EU-101, Fusion proteins and fragments, derivatives, variants, biosimilars and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and an OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein the one The or more TNFRSF agonists comprise 4-1BB agonists.

In some embodiments, the first expansion medium is referred to as "CM" (short for medium). In some embodiments, it is referred to as CM1 (Medium 1). In some embodiments, the CM consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL Gentamycin. In the example of initiating culture in a gas permeable bottle (eg, G-REX10; Wilson Wolf Manufacturing, New Brighton, MN) with a capacity of 40 mL and a gas permeable silicon ^{bottom of} 10 cm (Fig. 1), each bottle was loaded with 10-40 x ¹⁰⁶ live tumor digest cells or 5-30 tumor fragments in 10-40 mL CM with IL-2. Both G-REX10 and 24-well plates were cultured in a humidified incubator at 37°C, 5% CO ₂ and 5 days after the initiation of culture, half of the medium was removed and replaced with fresh CM and IL-2, and the After day 5, half of the medium was changed every 2-3 days. In some embodiments, CM is CM1 as described in the Examples, see Example 1. In some embodiments, the first expansion occurs in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial or first cell culture medium comprises IL-2.

In some embodiments, the first amplification (including procedures such as those described in FIG. 1 or Step B of FIG. 8 , which may include what is sometimes referred to as pre-REP) is shortened to 3- 14 days, as discussed in the Examples and Figures. In some embodiments, the first amplification (including a process such as that described in Figure 1 or Step B of Figure 8, which may include a process sometimes referred to as pre-REP) is shortened to 7 to 14 days, as shown in the Examples Discussion and shown in FIG. 4 and FIG. 5 , and includes, for example, the amplification described in FIG. 1 or Step B of FIG. 8 . In some embodiments, the first amplification of step B is shortened to 10-14 days. In some embodiments, the first amplification is shortened to 11 days, as discussed in, for example, the amplification described in FIG. 1 or step B of FIG. 8 .

In some embodiments, the first TIL expansion can be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days days or 14 days. In some embodiments, the first TIL expansion can be performed for 1 to 14 days. In some embodiments, the first TIL expansion can be performed for 2 days to 14 days. In some embodiments, the first TIL expansion can be performed for 3 days to 14 days. In some embodiments, the first TIL expansion can be performed for 4 days to 14 days. In some embodiments, the first TIL expansion can be performed for 5 days to 14 days. In some embodiments, the first TIL expansion can be performed for 6 days to 14 days. In some embodiments, the first TIL expansion can be performed for 7 days to 14 days. In some embodiments, the first TIL expansion can be performed for 8 to 14 days. In some embodiments, the first TIL expansion can be performed for 9 days to 14 days. In some embodiments, the first TIL expansion can be performed for 10 to 14 days. In some embodiments, the first TIL expansion can be performed for 11 days to 14 days. In some embodiments, the first TIL expansion can be performed for 12 to 14 days. In some embodiments, the first TIL expansion can be performed for 13 to 14 days. In some embodiments, the first TIL expansion can be performed for 14 days. In some embodiments, the first TIL expansion can be performed for 1 to 11 days. In some embodiments, the first TIL expansion can be performed for 2 days to 11 days. In some embodiments, the first TIL expansion can be performed for 3 days to 11 days. In some embodiments, the first TIL expansion can be performed for 4 days to 11 days. In some embodiments, the first TIL expansion can be performed for 5 to 11 days. In some embodiments, the first TIL expansion can be performed for 6 days to 11 days. In some embodiments, the first TIL expansion can be performed for 7 days to 11 days. In some embodiments, the first TIL expansion can be performed for 8 to 11 days. In some embodiments, the first TIL expansion can be performed for 9 days to 11 days. In some embodiments, the first TIL expansion can be performed for 10 to 11 days. In some embodiments, the first TIL expansion can be performed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15 and/or IL-21 is employed as the combination during the first expansion. In some embodiments, IL-2, IL-7, IL-15, and/or IL-21 may be included during the first amplification, including, for example, during step B of the process according to FIG. 1 or FIG. 8 and described herein. and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is employed as the combination during the first expansion. In some embodiments, IL-2, IL-15, and IL-21 , and any combination thereof, can be included during Step B of the process according to FIG. 1 or FIG. 8 and as described herein.

In some embodiments, the first amplification (including a process called pre-REP; eg, step B according to FIG. 1 or FIG. 8 ) process is shortened to 3 to 14 days, as discussed in the examples and figures. In some embodiments, the first amplification of Step B is shortened to 7 to 14 days. In some embodiments, the first amplification of Step B is shortened to 10 to 14 days. In some embodiments, the first expansion is shortened to 11 days.

In some embodiments, the first amplification is performed in a closed system bioreactor, eg according to step B of FIG. 1 or FIG. 8 . In some embodiments, TIL expansion as described herein is performed using a closed system. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor. 1. Cytokines and other additives

The expansion methods described herein typically use media with high doses of cytokines, particularly IL-2, as known in the art.

Alternatively, rapid and/or secondary expansion of TILs is also possible using a combination of cytokines, as described in US Patent Application Publication No. US 2017/0107490 A1 using IL-2, IL-15 and a combination of two or more than two of IL-21, the disclosure of this case is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, or IL-15 and IL-21, the latter being in many embodiments have a specific purpose. The use of a combination of cytokines is particularly advantageous for the generation of lymphocytes, and especially T cells as described therein.

In some embodiments, step B may also include adding OKT-3 antibody or murozumab to the culture medium, as described elsewhere herein. In some embodiments, step B can also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B can also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma, can be used in the culture medium during step B Agonists, such as thiazolidinedione compounds, are described in US Patent Application Publication No. US 2019/0307796 A1, the disclosure of which is incorporated herein by reference.

In some embodiments, step B may also include adding protein kinase B (AKT) inhibitor (AKTi) to the culture medium. In some embodiments, the TIL population is cultured in a medium containing an AKT inhibitor to obtain a CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population. In some embodiments, the AKT inhibitor is selected from the group consisting of pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, Oridonin, gossinthin, tenoride, isoliquiritigenin, chrysanthemum, honokiol and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is pataxerti. In some embodiments, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, About 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM or about 5 μM in the culture medium of pataxerti Cultivate TIL populations. C. Step C : Transition from First Amplification to Second Amplification

In some cases, the subject TIL population obtained from the first expansion, including, for example, the TIL population obtained from Step B as shown in, for example, Figure 1 or Figure 8, can be immediately cryopreserved using the protocols discussed below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, can be subjected to a second expansion (which may include expansion sometimes referred to as REP) and then cryopreserved as discussed below. Similarly, where genetically modified TILs are used in therapy, either the first TIL population (sometimes referred to as the subject TIL population) or the second TIL population (which in some embodiments may include a population known as the REP TIL population) population) can be genetically modified prior to amplification or after a first amplification and before a second amplification for appropriate therapy.

In some embodiments, TILs obtained from the first amplification (eg, from step B as indicated in Figure 1 or Figure 8) are stored until phenotyped for selection. In some embodiments, TILs obtained from the first amplification (eg, from step B as indicated in Figure 1 or Figure 8) are not stored and proceed directly to the second amplification. In some embodiments, the TILs obtained from the first expansion are not cryopreserved after the first expansion and before the second expansion. In some embodiments, the transition from the first amplification to the second amplification occurs about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, Occurs in 12, 13 or 14 days. In some embodiments, the transition from the first amplification to the second amplification occurs about 3 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs about 4 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs about 4 days to 10 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs about 7 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs about 14 days after fragmentation occurs.

In some embodiments, the transition from the first amplification to the second amplification occurs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days after fragmentation occurs , 10, 11, 12, 13 or 14 days. In some embodiments, the transition from the first amplification to the second amplification occurs 1 to 14 days after fragmentation occurs. In some embodiments, the first TIL expansion can be performed for 2 days to 14 days. In some embodiments, the transition from the first amplification to the second amplification occurs between 3 days and 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 4 to 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 5 and 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 6 to 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 7 to 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 8 to 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 9 and 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 10 to 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 11 and 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 12 to 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 13 to 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 14 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 1 day and 11 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 2 days and 11 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 3 days and 11 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 4 days and 11 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 5 and 11 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 6 to 11 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 7 and 11 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 8 to 11 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 9 and 11 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 10 to 11 days after fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 11 days after fragmentation occurs.

In some embodiments, the TILs are not stored after the first amplification and prior to the rapid second amplification, and the TILs are directly subjected to the second amplification (e.g., in some embodiments, as shown in FIG. 1 and/or FIG. 8 No storage was performed during the transition from step B to step D shown in ). In some embodiments, the transformation occurs in a closed system as described herein. In some embodiments, TILs from the first expansion (the second population of TILs) are directly subjected to the second expansion without a transition period.

In some embodiments, the transition from the first amplification to the second amplification (eg step C according to Figure 1 or Figure 8) is performed in a closed system bioreactor. In some embodiments, TIL expansion as described herein is performed using a closed system. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed system bioreactor is a single bioreactor. D. Step D : Second Amplification

In some embodiments, the population of TIL cells is expanded in number after collection and initial bulk processing, eg, after steps A and B, and the transition is referred to as step C, as indicated in FIG. 1 or FIG. 8 . This further amplification, referred to herein as the second amplification, may include an amplification process commonly referred to in the art as the Rapid Amplification Process (REP); and as indicated in step D of FIG. 1 or FIG. 8 the process. The second expansion is generally accomplished in a gas-permeable vessel using a culture medium containing various components, including feeder cells, a source of cytokines, and an anti-CD3 antibody.

In some embodiments, the second amplification or second TIL amplification (which may include amplification sometimes referred to as REP; and the process indicated in step D of FIG. 1 ) can be performed using Any known TIL bottle or container. In some embodiments, the second TIL expansion can be performed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second TIL expansion can be performed for about 7 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 8 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 9 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 10 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 11 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 12 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 13 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 14 days.

In some embodiments, the second amplification can be performed in a gas-permeable vessel using the methods of the disclosure (including, for example, amplification known as REP; and the process indicated in Figure 1 or Step D of Figure 8). For example, TILs can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulators can include, for example, anti-CD3 antibodies such as about 30 ng/ml OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Raritan, NJ or Auburn, CA Miltenyi Biotechnology, Inc., San Diego, California) or UHCT-1 (available from BioLegend, San Diego, CA, USA). TILs can be amplified to induce further stimulation of TILs in vitro by including one or more cancer antigens (including antigenic portions thereof such as epitopes) during a second expansion, optionally in T cell growth factors ( Expressed from a carrier, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) in the presence of IL-2 or IL-15, such as 300 IU/mL, as appropriate or gpl 00:209-217(210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same cancer antigen pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of the second amplification. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL , about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL- 2. In some embodiments, the cell culture medium comprises 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL , 7000 to 8000 IU/mL, or 8000 IU/mL IL-2.

In some embodiments, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL , about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 µg/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium does not contain an OKT-3 antibody. In some embodiments, the OKT-3 antibody is murozumab.

In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: Urelumab, Utumumab, EU-101, Fusion proteins and fragments, derivatives, variants, biosimilars and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and an OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein the one The or more TNFRSF agonists comprise 4-1BB agonists.

In some embodiments, a combination of IL-2, IL-7, IL-15 and/or IL-21 is employed as the combination during the second expansion. In some embodiments, IL-2, IL-7, IL-15, and/or IL-21 may be included during the second amplification, including, for example, during step D according to FIG. 1 or FIG. 8 and described herein. and any combination thereof. In some embodiments, a combination of IL-2, IL-15 and IL-21 is employed as the combination during the second expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, can be included during the process according to Figure 1 or Figure 8 and step D as described herein.

In some embodiments, the second expansion can be performed in supplemented cell culture medium comprising IL-2, OKT-3, antigen presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3 and antigen presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen presenting cells (APCs; also known as antigen presenting feeder cells). In some embodiments, the second expansion occurs in cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (ie, antigen-presenting cells).

In some embodiments, the second expansion medium comprises about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 200 IU/mL IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15.

In some embodiments, the second expansion medium comprises about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21 twenty one. In some embodiments, the second expansion medium comprises about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 2 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TIL to PBMC and/or antigen presenting cells in the rapid expansion and/or the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125 , about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375 , about 1:400 or about 1:500. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1:100 and 1:200.

In some embodiments, REP and/or secondary expansion is performed in culture flasks, wherein bulk TIL is mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/ mL IL-2 was mixed in 150 mL medium. Perform media changes (typically 2/3 media replacement via aspiration with fresh media) until cells are transferred to an alternate growth chamber. Alternative growth chambers include G-REX flasks and gas permeable containers, as discussed more fully below.

In some embodiments, the second amplification (which may include a process known as the REP process) is shortened to 7 to 14 days, as discussed in the Examples and Figures. In some embodiments, the second expansion is shortened to 11 days.

In some embodiments, REP and/or the second amplification can use previously described T-175 culture flasks and air-permeable bags (Tran et al., " J. Immunother. )" 2008, 31, 742-51 ; Dudley et al., Journal of Immunotherapy 2003, 26, 332-42) or gas-permeable Petri dishes (G-REX bottles). In some embodiments, the second expansion (including expansion called rapid expansion) is performed in T-175 culture flasks, and about 1 x ¹⁰⁶ TILs suspended in 150 mL of medium can be added to Each T-175 bottle. TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU/mL IL-2 and 30 ng/ml anti-CD3. T-175 flasks can be incubated at 37°C in 5% CO ₂ . Half of the medium can be replaced on day 5 with 50/50 medium with 3000 IU/mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks can be combined in a 3 L bag and 300 mL of 5% human AB serum and 3000 IU/mL IL-2 AIM V was added to 300 mL of TIL suspension. The number of cells in each bag was counted daily or every two days, and fresh medium was added to maintain cell counts between 0.5 and 2.0 x ¹⁰⁶ cells/ml.

In some embodiments, the second amplification (which may include the amplification referred to as REP, and those mentioned in step D of FIG. 1 or FIG. 8 ) can be performed in a 500 mL capacity chamber with a 100 cm gas Silicon-bottomed gas-permeable culture flasks (G-REX-100, available from Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA), 5×10 ⁶ or 10×10 ⁶ TILs Can be cultured with PBMC in 400 mL of 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2 and 30 ng/mL anti-CD3 (OKT3). G-REX-100 flasks can be incubated at 37°C in 5% CO ₂ . On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 xg) for 10 minutes. The TIL pellet can be resuspended with 150 mL of fresh medium containing 5% human AB serum, 3000 IU/mL IL-2, and added back to the original G-REX-100 culture flask. When TILs are continuously expanded in G-REX 100 flasks, on day 7, TILs in each G-REX-100 can be suspended in the 300 mL medium present in each flask, and the cell suspension can be divided into Three 100 mL aliquots from three G-REX-100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 can be added to each flask. G-REX-100 flasks can be incubated at 37°C, 5% CO2 and after 4 days, 150 mL of AIM-V with 3000 IU/mL IL-2 can be added to each G-REX-100 flask . Cells can be harvested on day 14 of culture.

In some embodiments, the second expansion (including expansion called REP) is performed in culture flasks in which host TILs are mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti- CD3 antibody and 3000 IU/mL IL-2 were mixed in 150 mL medium. In some embodiments, the medium is replaced until the cells are transferred to an alternate growth chamber. In some embodiments, 2/3 of the medium was replaced with fresh medium by aspiration. In some embodiments, alternative growth chamber chambers include G-REX flasks and gas permeable containers, as discussed more fully below.

In some embodiments, a second amplification is performed (comprising amplification referred to as REP), and further comprising the step of selecting TILs with superior heterotumor reactivity. Any selection method known in the art may be used. For example, the methods described in US Patent Application Publication No. 2016/0010058 A1 , the disclosure of which is incorporated herein by reference, can be used to select TILs with superior tumor reactivity.

Optionally, cell viability analysis can be performed using standard assays known in the art following secondary amplification, including that referred to as REP amplification. For example, a trypan blue exclusion assay, which selectively marks dead cells and allows assessment of viability, can be performed on bulk TIL samples. In some embodiments, TIL samples can be counted and assessed for viability using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

In some embodiments, secondary expansion of TILs, including expansion termed REP, can be performed using T-175 flasks and gas permeable bags as previously described (Tran et al., 2008 , Journal of Immunotherapy, 31 , 742-751, and Dudley et al., 2003 , "Journal of Immunotherapy", 26 , 332-342) or gas-permeable G-REX culture flasks. In some embodiments, bottles are used for the second amplification. In some embodiments, the second expansion is performed using a gas permeable G-REX flask. In some embodiments, the second expansion is performed in T-175 flasks, and about 1 x ¹⁰⁶ TILs are suspended in about 150 mL of medium and added to each T-175 flask. TILs were cultured with irradiated (50 Gy) allogeneic PBMCs as "feeder" cells at a ratio of 1:100 and cells were incubated in CM supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3 and AIM - Cultured in a 1:1 mixture (50/50 medium) of V medium. T-175 flasks were incubated at 37°C in 5% CO ₂ . In some embodiments, half of the medium is replaced on day 5 with 50/50 medium containing 3000 IU/mL IL-2. In some embodiments, on day 7, cells from 2 T-175 flasks were pooled in a 3 L bag and 300 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 Add to 300 mL TIL suspension. The number of cells in each bag can be counted daily or every two days, and fresh medium can be added to maintain cell counts between about 0.5 and about 2.0 x ¹⁰⁶ cells/mL.

In some embodiments, the second expansion (including that referred to as REP) is performed in a 500 mL culture flask with approximately 5 µL of a 100 ^cm gas permeable silicon bottom (G-REX-100, Wilson Wolf). × ¹⁰⁶ or 10× ¹⁰⁶ TILs were cultured with irradiated allogeneic PBMCs at a ratio of 1:100 in 400 mL 50/50 medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3 . G-REX-100 flasks were incubated at 37°C in 5% CO ₂ . In some embodiments, on day 5, 250 mL of supernatant is removed and placed into a centrifuge bottle and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellets can then be resuspended with 150 mL of fresh 50/50 medium with 3000 IU/mL IL-2 and added back to the original G-REX-100 culture flask. In the example where TILs were continuously expanded in G-REX-100 culture flasks, on day 7, TILs in each G-REX-100 were suspended in 300 mL of medium present in each culture flask, and the cells were suspended The solution was divided into three 100 mL aliquots that could be used to inoculate 3 G-REX-100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 was added to each flask. G-REX-100 flasks were incubated at 37°C in 5% _CO and after 4 days, 150 mL of AIM-V with 3000 IU/mL IL-2 was added to each G-REX-100 flask . Cells can be harvested on day 14 of culture.

The diverse antigen receptors of T and B lymphocytes are generated by somatic recombination of a limited but large number of gene segments. These gene segments: V (variable region), D (diversity region), J (junction region) and C (constant region) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCR). The present invention provides a method for generating TILs that exhibit and increase the diversity of the T cell repertoire. In some embodiments, TILs obtained by the methods of the invention exhibit increased T cell repertoire diversity. In some embodiments, the TILs obtained in the second expansion exhibit increased T cell repertoire diversity. In some embodiments, increasing diversity is increasing immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, present in immunoglobulin heavy chains. In some embodiments, the diversity is present in immunoglobulins, in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and/or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, TCRab (ie, TCRα/β) expression is increased.

In some embodiments, the second expansion medium (eg, sometimes referred to as CM2 or second cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in more detail below.

In some embodiments, the medium used in the expansion processes disclosed herein is a serum-free medium or a defined medium. In some embodiments, serum-free or defined media comprise basal cell culture media and serum supplements and/or serum substitutes. In some embodiments, serum-free or defined media are used to prevent and/or reduce experimental variation in part due to batch-to-batch variation of serum-containing media.

In some embodiments, serum-free or defined media comprise basal cell culture media and serum supplements and/or serum substitutes. In some embodiments, basal cell culture media include, but are not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T cell expansion Xeno-free medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI Growth Medium, and Ischoff's Modified Dulbecco's Medium.

In some embodiments, serum supplements or serum replacements include, but are not limited to, one or more of: CTS™ OpTmizer T Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagens Protein precursors, one or more antibiotics and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L - Phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine , reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and trace elements containing Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ , Ni ²⁺ , Rb ⁺ , Sn ²⁺ and Zr ⁴⁺ compounds. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some embodiments, CTS™ OpTmizer™ T Cell Immune Cell Serum Replacement is used with conventional growth media including, but not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dahl Burke's medium.

In some embodiments, the total serum replacement concentration (vol %) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, based on the total volume of the serum-free or defined medium. 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In some embodiments, the total serum replacement concentration is about 3% of the total volume of serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of serum-free or defined medium.

In some embodiments, the serum-free or defined medium is a CTS™ OpTmizer™ T cell expansion SFM (Thermo Fisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 µM.

In some embodiments, the defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (Thermo Fisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L - Glutamine. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 6000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 3000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 µM.

In some embodiments, serum-free medium or synthetic medium is supplemented with a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM or 4 mM to about 5 mM glutamine (ie, GlutaMAX®). In some embodiments, the serum-free medium or synthetic medium is supplemented with glutamine (ie, GlutaMAX®) at a concentration of about 2 mM.

In some embodiments, serum-free medium or defined medium is supplemented with a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM , 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or About 65 mM of 2-mercaptoethanol. In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the medium is 55 µM.

In some embodiments, defined media as described in International PCT Publication No. WO/1998/030679, incorporated herein by reference, find use in the present invention. In this publication, a serum-free eukaryotic cell culture medium is described. Serum-free eukaryotic cell culture media include basal cell culture media supplemented with serum-free supplements capable of supporting the growth of cells in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises one or more components selected from the group consisting of, or is obtained by combining one or more components selected from the group consisting of: one or more albumins or albumin substitutes one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, One or more trace elements and one or more antibiotics. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin Ferritin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L - Phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine , reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and trace elements containing Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ , Ni ²⁺ , Rb ⁺ , Sn ²⁺ and Zr ⁴⁺ compounds. In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640 , F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dulbecco's Medium.

In some embodiments, it is determined that the concentration of glycine in the medium is in the range of about 5 to 200 mg/L, the concentration of L-histidine is in the range of about 5 to 250 mg/L, and the concentration of L-isoleucine is The concentration of L-methionine is about 5 to 300 mg/L, the concentration of L-methionine is about 5 to 200 mg/L, the concentration of L-phenylalanine is about 5 to 400 mg/L, and the concentration of L-proline is about 1 to 1000 mg/L, the concentration of L-hydroxyproline is about 1 to 45 mg/L, the concentration of L-serine is about 1 to 250 mg/L, and the concentration of L-threonine is about 10 to 500 mg/L, the concentration of L-tryptophan is about 2 to 110 mg/L, the concentration of L-tyrosine is about 3 to 175 mg/L, and the concentration of L-valine is about 5 to 500 mg/L mg/L, the concentration of thiamine is about 1 to 20 mg/L, the concentration of reduced glutathione is about 1 to 20 mg/L, and the concentration of L-ascorbic acid-2-phosphate is about 1 to 200 mg /L, the concentration of iron-saturated transferrin is about 1 to 50 mg/L, the concentration of insulin is about 1 to 100 mg/L, the concentration of sodium selenite is about 0.000001 to 0.0001 mg/L, and albumin ( For example, the concentration of AlbuMAX® I) is about 5000 to 50,000 mg/L.

In some embodiments, the non-trace element fraction of the defined medium is present in the concentration ranges listed in the column heading "Concentration Ranges in 1X Medium" in Table 4. In other embodiments, the non-trace element components of the defined medium are present at the final concentrations listed in Table 4 in the column headed "Preferred Embodiments of 1X Medium". In other embodiments, the defined medium is a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trivial fraction ingredients of the type and concentration listed in the column heading "Preferred Embodiments of Supplements" in Table 4.

In some embodiments, the osmolarity of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmolarity is between about 280 and 310 mOsmol. In some embodiments, defined medium is supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. Defined media can be further supplemented with L-glutamine (at a final concentration of approximately 2 mM), one or more antibiotics, non-essential amino acids (NEAA; at a final concentration of approximately 100 μM), 2-mercaptoethanol (at a final concentration of about 100 μM).

In some embodiments, defined media as described in Smith et al., Clin Transl Immunology 4(1) 2015 (doi: 10.1038/cti.2014.31 ) can be used in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as basal cell culture medium supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

In some embodiments, the cell culture medium in the first and/or second gas permeable container is unfiltered. The use of unfiltered cell culture media simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second gas-permeable container lacks β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some embodiments, the second amplification is performed in a closed system bioreactor, such as step D according to FIG. 1 or FIG. 8 . In some embodiments, TIL expansion as described herein is performed using a closed system. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, the step of rapid or secondary expansion is divided into multiple steps to achieve vertical scaling up of the culture scale by (a) scaling up in the first vessel (eg, G-REX- 100 MCS vessel) for rapid or second expansion by culturing TILs in small scale cultures for a period of about 3 days to 7 days; and then (b) effectuating transfer of TILs in small scale cultures to larger vessels than the first vessel A second vessel (eg, a G-REX-500-MCS vessel) and culture the TILs from the small scale culture in the larger scale culture in the second vessel for a period of about 4 to 7 days.

In some embodiments, the step of rapid or secondary expansion is divided into multiple steps to achieve lateral scale-up of culture by: (a) performing rapid or second expansion in the first small-scale culture of culturing T cells for a period of about 3 to 7 days; and then (b) transferring and distributing T cells from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers of the same size as the first container, wherein In each second vessel, the portion of T cells from the first mini-culture transferred to such second vessel is cultured in the second mini-culture for a period of about 4 to 7 days.

In some embodiments, the first small-scale TIL culture is partitioned into a plurality of about 2 to 5 TIL subpopulations.

In some embodiments, the step of rapid or second expansion is divided into multiple steps to achieve horizontal and vertical scale-up of the culture by: (a) - 100 MCS containers in which TILs are cultured in small-scale cultures for a period of about 3 to 7 days to perform rapid or secondary expansion; and then (b) transfer and distribute TILs from the small-scale cultures to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers larger in size than the first, such as G-REX - In 500 MCS containers, wherein in each second container, the TIL fraction from the small scale culture transferred to such second container is grown in the larger scale culture for a period of about 4 to 7 days.

In some embodiments, the step of rapid or second expansion is divided into multiple steps to achieve scale-up and scale-up of the culture by: (a) through the first container (such as G-REX- 100 MCS vessels) to culture TILs in small scale cultures for a period of about 5 days for rapid or second expansion; and then (b) effect transfer and distribution of TILs from small scale cultures to 2, 3 or 4 In second containers (e.g., G-REX-500 MCS containers) larger in size than the first container, wherein in each second container, the fraction of TILs from small-scale cultures transferred to such second containers is on a larger scale The culture was cultured for a period of about 6 days.

In some embodiments, each second container comprises at least ¹⁰⁸ TILs at the time of rapid or second expansion. In some embodiments, each second container comprises at least 10 ⁸ TILs, at least 10 ⁹ TILs, or at least 10 ¹⁰ TILs at the time of rapid or second expansion. In an exemplary embodiment, each second container contains at least ¹⁰¹⁰ TILs.

In some embodiments, the first small-scale TIL culture is divided into subpopulations. In some embodiments, the first small-scale TIL culture is partitioned into a plurality of about 2 to 5 subpopulations. In some embodiments, the first small-scale TIL culture is partitioned into a plurality of about 2, 3, 4, or 5 subpopulations.

In some embodiments, the plurality of subpopulations comprise a therapeutically effective amount of TILs following completion of the rapid or second expansion. In some embodiments, following rapid or secondary expansion, one or more subpopulations of TILs are pooled together to produce a therapeutically effective amount of TILs. In some embodiments, each subpopulation of TILs comprises a therapeutically effective amount of TILs following rapid expansion.

In some embodiments, the rapid or second amplification is performed for a period of about 3 to 7 days before dividing into a plurality of steps. In some embodiments, seeding of the rapid or second amplification occurs at about day 3, day 4, day 5, day 6, or day 7 after initiation of the rapid or second amplification.

In some embodiments, splitting of the rapid or second amplification occurs on about day 7, day 8, day 9, day 10, day 1 after initiation of the first amplification (i.e., pre-REP amplification). Day 11, Day 12, Day 13, Day 14, Day 15 or Day 16, Day 17 or Day 18. In an exemplary embodiment, the fraction of rapid or second amplification occurs about day 16 after initiation of the first amplification.

In some embodiments, the rapid or secondary expansion is further performed for a period of about 7 to 11 days after splitting. In some embodiments, the rapid or secondary expansion is further performed for a period of about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days after seeding.

In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting comprises the same components as the cell culture medium used for rapid or second expansion after splitting. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting comprises different components than the cell culture medium used for rapid or second expansion after splitting.

In some embodiments, the cell culture medium for rapid or secondary expansion prior to splitting comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium for rapid or secondary expansion prior to splitting comprises IL-2, OKT-3 and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion prior to splitting comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is supplemented in the first expansion with fresh medium comprising IL-2, optionally OKT-3, and further optionally APC. Produced by cell culture medium. In some embodiments, the cell culture medium used for rapid or second expansion prior to splitting is produced by supplementing the cell culture medium in the first expansion with fresh medium comprising IL-2, OKT-3, and APC . In some embodiments, cell culture medium used for rapid or second expansion prior to splitting is performed by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, optionally OKT-3, and further optionally APC. produced by cell culture medium. In some embodiments, cell culture medium for rapid or second expansion prior to splitting is produced by replacing cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, OKT-3, and APC of.

In some embodiments, the cell culture medium for rapid or secondary expansion after splitting comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium for rapid or secondary expansion after splitting comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid or second expansion after splitting is by replacing the cell culture medium used for rapid or second expansion before splitting with fresh medium comprising IL-2 and optionally OKT-3 produced by cell culture medium. In some embodiments, the cell culture medium used for rapid or second expansion after splitting is obtained by replacing the cells used for rapid or second expansion before splitting with fresh medium comprising IL-2 and OKT-3 produced by the culture medium.

In some embodiments, rapid expansion of strains occurs in a closed system.

In some embodiments, scaling up the TIL culture vertically during the rapid or second expansion comprises adding fresh cell culture medium to the TIL culture (also referred to as feeding the TIL). In some embodiments, feeding comprises frequent addition of fresh cell culture medium to the TIL culture. In some embodiments, feeding comprises adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TIL via a constant flow. In some embodiments, an automated cell expansion system, such as Xuri W25, is used for rapid expansion and feeding. 1. Feeder cells and antigen-presenting cells

In some embodiments, the second amplification procedure described herein (e.g., including amplification such as described in FIG. 1 or step D of FIG. 8 and referred to as REP) is performed during REP TIL amplification and /or excess feeder cells are required during the second expansion. In many embodiments, the feeder cell line is peripheral blood mononuclear cells (PBMC) obtained from a standard whole blood unit of a healthy blood donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation.

In general, allogeneic PBMCs were not activated by irradiation or heat treatment, and were used in the REP procedure as described in the Examples, which provide an exemplary protocol for assessing the non-replication capacity of irradiated allogeneic PBMCs.

In some embodiments, if the total number of viable cells on day 14 is less than that placed in the culture on day 0 of REP and/or day 0 of the second expansion (i.e., the start date of the second expansion) Initial viable cell numbers, PBMC were considered replication-incompetent and acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on day 7 and day 14 is the same as that on day 0 of REP and/or day 0 of the second expansion (i.e., second Initiation date of expansion) compared to the initial number of viable cells placed in culture, the PBMC were considered replication-incompetent and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on day 7 and day 14 is the same as that on day 0 of REP and/or day 0 of the second expansion (i.e., second Initiation date of expansion) compared to the initial number of viable cells placed in culture, the PBMC were considered replication-incompetent and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5 to 60 ng/mL OKT3 antibody and 1000 to 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10 to 50 ng/mL OKT3 antibody and 2000 to 5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20 to 40 ng/mL OKT3 antibody and 2000 to 4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25 to 35 ng/mL OKT3 antibody and 2500 to 3500 IU/mL IL-2.

In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen presenting feeder cells in the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175 , about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400 or about 1:500 . In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion procedure described herein requires a ratio of about 2.5×10 ⁹ feeder cells:about 100×10 ⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of about 2.5×10 ⁹ feeder cells:about 50×10 ⁶ TILs. In other embodiments, the second expansion procedure described herein requires a ratio of about 2.5×10 ⁹ feeder cells:about 25×10 ⁶ TILs.

In some embodiments, the second expansion procedure described herein requires an excess of feeder cells during the second expansion. In many embodiments, the feeder cell line is peripheral blood mononuclear cells (PBMC) obtained from a standard whole blood unit of a healthy blood donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen presenting cells (aAPCs) are used instead of PBMCs.

In general, allogeneic PBMCs were inactivated by irradiation or heat treatment and used in the TIL expansion procedures described herein, including the exemplary procedures described in the Figures and Examples.

In some embodiments, artificial antigen presenting cells are used in place of or in combination with PBMCs in the second expansion. 2. Cytokines and other additives

The expansion methods described herein typically use media with high doses of cytokines, particularly IL-2, as known in the art.

Alternatively, rapid and/or secondary expansion of TILs is also possible using a combination of cytokines, as described in US Patent Application Publication No. US 2017/0107490 A1 using IL-2, IL-15 and a combination of two or more than two of IL-21, the disclosure of this case is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 as well as IL-2, IL-15 and IL-21, where the latter in many embodiments has a specific use. The use of a combination of cytokines is particularly advantageous for the generation of lymphocytes, and especially T cells as described therein.

In some embodiments, step D may also include adding OKT-3 antibody or murozumab to the culture medium, as described elsewhere herein. In some embodiments, step D can also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D can also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. In addition, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists, may be used in the culture medium during step D, Such as thiazolidinedione compounds, as described in US Patent Application Publication No. US 2019/0307796 A1, the disclosure of which is incorporated herein by reference.

In some embodiments, step D may also include adding protein kinase B (AKT) inhibitor (AKTi) to the culture medium. In some embodiments, the TIL population is cultured in a medium containing an AKT inhibitor to obtain a CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population. In some embodiments, the AKT inhibitor is selected from the group consisting of pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, Oridonin, gossinthin, tenoride, isoliquiritigenin, chrysanthemum, honokiol and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is pataxerti. In some embodiments, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, About 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM or about 5 μM in the culture medium of pataxerti Cultivate TIL populations. E. Step E : Collect TIL

After the second expansion step, the cells can be harvested. In some embodiments, TILs are collected after one, two, three, four or more amplification steps such as provided in FIG. 1 or FIG. 8 . In some embodiments, TILs are collected after two amplification steps such as provided in FIG. 1 or FIG. 8 .

TILs can be collected by any suitable and sterile means including, for example, centrifugation. Methods for collecting TILs are well known in the art and any such known methods may be used with the procedures of the present invention. In some embodiments, TILs are collected using an automated system.

Cell harvesters and/or cell processing systems are commercially available from a variety of sources including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell-based harvester can be used in the methods of the invention. In some embodiments, the cell harvester and/or cell processing system is a membrane-based cell harvester. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument manufactured by any supplier that pumps a solution containing cells through a membrane or filter (such as a spin membrane or spin filter) in a sterile and/or closed system environment Or devices that allow continuous flow and cell handling to remove supernatant or cell culture medium without clumping. In some embodiments, the cell harvester and/or cell processing system can perform cell isolation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed sterile system.

In some embodiments, the self-closed system bioreactor is collected, eg, according to step E of FIG. 1 or FIG. 8 . In some embodiments, TIL expansion as described herein is performed using a closed system. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, step E according to FIG. 1 or FIG. 8 is performed according to the processes described herein. In some embodiments, the closed system is accessed via a syringe under aseptic conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the Examples is used.

In some embodiments, TILs are collected according to the methods described in the Examples. In some embodiments, TILs are collected between day 1 and day 11, such as in the examples on day 11, using methods as described in the steps referred to herein. In some embodiments, TILs are collected between day 12 and day 24, such as in the examples on day 22, using methods as described in the steps referred to herein. In some embodiments, the invention provides for assessing or sorting the therapeutic TIL population or TIL composition described in the collection step described herein for: (i) CD39/CD69 double negative and/or CD39 ^LO / CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) a combination of (i) and (ii) TIL. Methods for sorting TILs for the following can be found in U.S. Application No. 2019/0212332: (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double gene knockout, or (iii) A combination of (i) and (ii), which is incorporated herein by reference. In some embodiments, using Zhang, X et al., "Surface Free Energy Activated High-Throughput Cell Sorting (Surface Free Energy Activated High-Throughput Cell Sorting)", "Analytical Chemistry (Analytical Chemistry)" (2014), 86: 9350-9355 for cell sorting. Example 15 and Figure 35 provide further examples of sorting schemes suitable for use in the methods of the invention. F. Step F : Final formulation and transfer to infusion container

After completion of Steps A to E as provided in the exemplary order shown in Figure 1 or Figure 8 and as outlined in detail above and herein, the cells are transferred to a container for administration to the patient, such as an infusion bag or sterile vial . In some embodiments, once therapeutically sufficient numbers of TILs are obtained using the expansion methods described above, they are transferred to containers for administration to a patient.

In some embodiments, TILs expanded using the APCs of the disclosure are administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TIL in a sterile buffer. TILs expanded using the PBMCs of the disclosure can be administered by any suitable route known in the art. In some embodiments, T cells are administered as a single intraarterial or intravenous infusion, which preferably lasts for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

In some embodiments, the present invention provides a method as described in any preceding paragraph, as applicable, such that (a) prior to sorting step (i), cells containing IL-2 and, optionally, a first antibiotic combination culturing the bulk TIL or first population of TILs in the tumor fragment or sample in culture medium to produce TILs released from the tumor fragment or sample, (ii) isolating at least a plurality of TILs released from the tumor fragment or sample from the tumor fragment or sample , to produce a tumor fragment or sample, a mixture of TIL remaining in the tumor fragment or sample, and any TIL released from the tumor fragment or sample and remaining with it after isolation, and (iii) combining the tumor fragment or sample, tumor fragment or residual TIL in the sample and any TIL released from the tumor fragment or sample and still with it after isolation to produce a digest of such mixture; (b) sorting the digest of the mixture to produce CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population, and (b) first amplification using the CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population. In some embodiments, at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 95%, 99% or higher percentage of TIL released from tumor fragments or samples is isolated from the tumor fragments or samples to produce a mixture.

In some embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the culturing step prior to the first expansion is performed for a period of about 1 day to about 3 days.

In some embodiments, the invention provides a method as described in any preceding paragraph, as applicable, modified such that the culturing step prior to the first expansion is performed for about 1, 2, 3, 4, 5, 6 or 7 day period.

In some embodiments, the invention provides a method as described in any preceding paragraph, as applicable, such that (a) the digestion, sorting, and first expansion steps comprise (i) culturing the tumor in IL-2-containing cell culture medium a bulk TIL or a first population of TILs in a fragment or sample to generate TILs released from a tumor fragment or sample, (ii) isolating at least a plurality of TILs released from a tumor fragment or sample from a tumor fragment or sample to generate a tumor fragment or sample, the mixture of TIL remaining in the tumor fragment or sample and any TIL released from the tumor fragment or sample and remaining with it after isolation, (iii) the tumor fragment or sample, the remaining TIL in the tumor fragment or sample Digestion of a mixture of TILs and any TILs released from the tumor fragment or sample and still with them after isolation to produce a digest of such mixture; and (iv) sorting the digest of the mixture to produce a CD39/CD69 dual negative and/or CD39 ^LO /CD69 ^LO TIL population, and (b) a second amplification using the CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population. In some embodiments, at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 95%, 99% or higher percentage of TILs released from tumor fragments or samples are isolated from the tumor fragments or samples to produce a mixture. IV. Gen 3 TIL Manufacturing Process

Without being bound by any particular theory, it is believed that an initial first expansion to initiate T cell activation followed by a rapid second expansion that enhances T cell activation, as described in the methods of the invention, allows preparations that retain "more The expanded T cells of the "young" phenotype are therefore expected to exhibit higher cytotoxicity against cancer cells than T cells expanded by other methods. In particular, it is believed that activation of T cells is initiated by exposure to anti-CD3 antibodies (e.g., OKT-3), IL-2, and optionally antigen-presenting cells (APCs) as taught by the methods of the invention and then by Enhanced by subsequent exposure to additional anti-CD-3 antibodies (e.g. OKT-3), IL-2, and APCs, which limit or avoid maturation of T cells in culture, resulting in a T cell population with a less mature phenotype , these T cells are less depleted due to culture expansion and exhibit higher cytotoxicity against cancer cells. In some embodiments, the step of rapid second expansion is divided into multiple steps to achieve vertical scaling up of the culture scale by (a) scaling up in the first container, such as G-REX-100 MCS A rapid second expansion is performed by culturing the T cells in small culture in the vessel for a period of about 3 to 4 days; and then (b) effectuating the transfer of the T cells in the small culture to a vessel larger than the first vessel T cells from the small culture are cultured in the larger culture in a second vessel, such as a G-REX-500 MCS vessel, for a period of about 4 to 7 days. In some embodiments, the step of rapid expansion is divided into multiple steps to achieve the lateral expansion of the culture scale (scaling out) by: (a) through the first container, such as G-REX-100 MCS A rapid second expansion is performed by culturing the T cells in the first small culture in the vessel for a period of about 3 to 4 days; and then (b) transferring and distributing the T cells from the first small culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers of the same size as the first container, wherein In each second vessel, the portion of T cells from the first mini-culture transferred to such second vessel is cultured in the second mini-culture for a period of about 4 to 7 days. In some embodiments, the step of rapid expansion is divided into multiple steps to achieve horizontal and vertical expansion of the culture scale by: A rapid second expansion is performed by culturing the T cells in the small culture for a period of about 3 to 4 days; and then (b) transfer and distribute the T cells from the small culture to at least 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers that are larger in size than the first container, such as G-REX-500MCS container , wherein in each second vessel, the fraction of T cells from the small-scale culture transferred to such second vessel is cultured in the larger-scale culture for a period of about 4 to 7 days. In some embodiments, the step of rapid expansion is divided into multiple steps to achieve horizontal and vertical expansion of the culture scale by: A rapid second expansion is performed in culturing T cells in small-scale cultures for a period of about 4 days; and then (b) T cells from the small-scale cultures are transferred and partitioned into 2, 3, or 4 cells that are larger in size than the first Larger second containers, such as G-REX-500 MCS containers, wherein in each second container, the fraction of T cells from small-scale cultures transferred to such second containers are cultured in larger-scale cultures A period of about 5 days.

In some embodiments, each second container comprises at least ¹⁰⁸ TILs at the time of rapid expansion. In some embodiments, each second container comprises at least ¹⁰⁸ TILs, at least ¹⁰⁹ TILs, or at least ¹⁰¹⁰ TILs at the time of rapid expansion. In an exemplary embodiment, each second container contains at least ¹⁰¹⁰ TILs.

In some embodiments, the first small-scale TIL culture is divided into subpopulations. In some embodiments, the first small-scale TIL culture is partitioned into a plurality of about 2 to 5 subpopulations. In some embodiments, the first small-scale TIL culture is partitioned into a plurality of about 2, 3, 4, or 5 subpopulations.

In some embodiments, the plurality of subpopulations comprise a therapeutically effective amount of TILs following rapid expansion. In some embodiments, after rapid expansion is complete, one or more subpopulations of TILs are pooled together to produce a therapeutically effective amount of TILs. In some embodiments, each subpopulation of TILs comprises a therapeutically effective amount of TILs following rapid expansion.

In some embodiments, rapid amplification is performed for a period of about 1 to 5 days before breaking into a plurality of steps. In some embodiments, the splitting of rapid expansion occurs at about day 1, day 2, day 3, day 4, or day 5 after initiation of rapid expansion.

In some embodiments, seeding of rapid amplification occurs on about day 8, day 9, day 10, day 11, day 12, or Day 13. In an exemplary embodiment, the splitting of the rapid expansion occurs about day 10 after initiation of the initial first expansion. In another exemplary embodiment, the division of rapid amplification occurs at about day 11 after initiation of the initial first amplification.

In some embodiments, after the step, rapid amplification is further performed for a period of about 4 to 11 days. In some embodiments, the rapid amplification is further performed for a period of about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days after stepping.

In some embodiments, the cell culture medium used for rapid expansion prior to step-up comprises the same components as the cell culture medium used for post-step rapid expansion. In some embodiments, the cell culture medium used for pre-step rapid expansion comprises different components than the cell culture medium used for post-step rapid expansion.

In some embodiments, the cell culture medium used for rapid expansion prior to the step comprises IL-2, optionally OKT-3, and further optionally comprises APC. In some embodiments, the cell culture medium used for rapid expansion prior to fractionation comprises IL-2, OKT-3 and further optionally comprises APC. In some embodiments, the cell culture medium used for rapid expansion prior to fractionation comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid expansion prior to the step is obtained by supplementing the cell culture medium in the first expansion with fresh medium comprising IL-2, optionally OKT-3, and further optionally APCs. produced by cell culture medium. In some embodiments, the cell culture medium used for rapid expansion prior to the step is generated by supplementing the cell culture medium in the first expansion with fresh medium comprising IL-2, OKT-3, and APC. In some embodiments, the cell culture medium used for rapid expansion prior to step-up is obtained by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, optionally OKT-3, and further optionally APCs. produced by cell culture medium. In some embodiments, the cell culture medium used for the rapid expansion prior to the step is generated by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium for step-by-step rapid expansion comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium for step-by-step rapid expansion comprises IL-2 and OKT-3. In some embodiments, cell culture medium for post-step rapid expansion is generated by replacing cell culture medium for pre-step rapid expansion with fresh medium comprising IL-2 and optionally OKT-3 . In some embodiments, cell culture medium for post-step rapid expansion is generated by replacing cell culture medium for pre-step rapid expansion with fresh cell culture medium comprising IL-2 and OKT-3.

In some embodiments, rapid expansion of strains occurs in a closed system.

In some embodiments, the vertical scale-up of the TIL culture during rapid expansion comprises adding fresh cell culture medium to the TIL culture (also referred to as feeding the TIL). In some embodiments, feeding comprises frequent addition of fresh cell culture medium to the TIL culture. In some embodiments, feeding comprises adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TIL via a constant flow. In some embodiments, an automated cell expansion system, such as Xuri W25, is used for rapid expansion and feeding.

In some embodiments, the rapid second expansion is performed after T cell activation by the initial first expansion begins to decrease, slow down, decline or resolve.

In some instances, the rapid second expansion is when T cell activation by the initial first expansion has been reduced by at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% followed.

In some embodiments, the rapid second expansion is performed after the T cell activation achieved by the initial first expansion has been reduced by a percentage in the range of just or about 1% to 100%.

In some embodiments, the rapid second expansion is when T cell activation by the initial first expansion has been reduced by just or about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

In some embodiments, the rapid second expansion is when T cell activation by the initial first expansion has been reduced by at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% followed.

In some cases, the rapid second expansion is when T cell activation by the initial first expansion has been reduced by just or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% followed.

In some embodiments, the reduction in T cell activation by the initial first expansion is determined by a reduction in the amount of interferon gamma released by the T cells in response to antigenic stimulation.

In some embodiments, the initial first expansion of T cells is performed over a period of at most exactly or about 7 days or about 8 days.

In some embodiments, the initial first expansion of T cells is performed over a period of at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or 8 days.

In some embodiments, the initial first expansion of T cells is performed over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

In some embodiments, the rapid second expansion of T cells is performed over a period of at most exactly or about 11 days.

In some embodiments, the rapid second expansion of T cells is at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or within 11 days.

In some embodiments, the rapid second expansion of T cells is over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days within.

In some embodiments, the initial first expansion of T cells is performed within a period of exactly or about 1 day to exactly or about 7 days and the rapid second expansion of T cells is between exactly or about 1 day to exactly Or within a period of approximately 11 days.

In some embodiments, the initial first expansion of T cells occurs over a period of at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days and the T cells The rapid second amplification is performed over a period of at most exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.

In some embodiments, the initial first expansion of T cells is performed within a period of exactly or about 1 day to exactly or about 8 days and the rapid second expansion of T cells is between exactly or about 1 day to exactly Or within a period of approximately 9 days.

In some embodiments, the initial first expansion of T cells is performed over a period of 8 days and the rapid second expansion of T cells is performed over a period of 9 days.

In some embodiments, the initial first expansion of T cells is performed within a period of exactly at or about 1 day to at or about 7 days and the rapid second expansion of T cells is at or about 1 day to at or about 7 days Or within a period of about 9 days.

In some embodiments, the initial first expansion of T cells is performed over a period of 7 days and the rapid second expansion of T cells is performed over a period of 9 days.

In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs).

In some embodiments, the T cells are myeloid infiltrating lymphocytes (MIL).

In some embodiments, the T cells are peripheral blood lymphocytes (PBL).

In some embodiments, T cells are obtained from a donor with cancer.

In some embodiments, the T cells are TILs obtained from tumors resected from patients with cancer.

In some embodiments, the T cells are MIL obtained from the bone marrow of a patient suffering from a hematological malignancy.

In some embodiments, the T cells are PBLs obtained from peripheral blood mononuclear cells (PBMCs) of a donor. In some embodiments, the donor has cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, Cancers caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancers caused by human papillomavirus , head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor suffers from a hematological malignancy.

In certain aspects of the invention, immune effector cells (eg, T cells) can be obtained from blood units collected from an individual using any number of techniques known to those skilled in the art, such as FICOLL isolation. In a preferred aspect, cells from the circulating blood of the individual are obtained by apheresis. The apheresis product usually contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one aspect, cells collected by apheresis can be washed to remove the plasma fraction and the cells are optionally placed in an appropriate buffer or culture medium for subsequent processing steps. In some embodiments, the cell line is washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution is deficient in calcium, and may be deficient in magnesium or may be deficient in many, if not all, divalent cations. In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and elutriating depleted monocytes, eg, by centrifugation through a PERCOLL gradient or by countercurrent centrifugation.

In some embodiments, the T cells are PBLs isolated from whole blood or lymphocyte-rich apheresis product of a donor. In some embodiments, the donor has cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, Cancers caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancers caused by human papillomavirus , head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor suffers from a hematological malignancy. In some embodiments, the PBL is removed by using positive or negative selection methods, i.e., using T cell phenotype markers (such as CD3+ CD45+), or removing non-T cell phenotype cells to leave PBL , from whole blood or lymphocyte-rich apheresis products. In other embodiments, PBLs are isolated by gradient centrifugation. After isolation of PBLs from donor tissue, initial first expansion of PBLs can be performed according to the initial first expansion step of any of the methods described herein, by adding a suitable number of isolated PBLs (in some embodiments, about 1× 10 ⁷ PBLs) were inoculated into the initial first expansion culture to start.

An exemplary TIL process called Process 3 (also referred to herein as Gen 3) that incorporates some of these features is depicted in FIG. 8 (in particular, for example, FIG. 8B and/or FIG. 8C and/or FIG. and/or FIG. 8F and/or FIG. 8G ), and some advantages of this embodiment of the invention compared to Gen 2 are described in FIG. 1 , FIG. 2 , FIG. 8 , FIG. 30 and FIG. And/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G). Embodiments of Gen 3 are shown in FIG. 1 , FIG. 8 and FIG. 30 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8G). Method 2A or Gen 2 or Gen 2A is also described in US Patent Publication No. 2018/0280436, which is incorporated herein by reference in its entirety. The Gen 3 procedure is also described in International Patent Publication WO 2020/096988.

As discussed and generally outlined herein, TILs are taken from patient samples and manipulated using a TIL expansion process described herein and referred to as Gen 3 to expand their number prior to transplantation into the patient. In some embodiments, TILs can optionally be genetically manipulated as discussed below. In some embodiments, TILs can be cryopreserved either before or after expansion. After thawing, it can also be restimulated to increase its metabolism prior to infusion into a patient.

In some embodiments, the initial first amplification (including a procedure referred to herein as Pre-Rapid Amplification (Pre-REP), and Figure 8 (in particular, such as Figure 8A and/or Figure 8B and/or Figure 8C and/or or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. The program (REP) procedure, and shown in FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) is The procedure of Step D) was shortened to 1 to 9 days, as discussed in detail below and in the Examples and Figures. In some embodiments, the initial first amplification (including a procedure referred to herein as Pre-Rapid Amplification (Pre-REP), and Figure 8 (in particular, such as Figure 8A and/or Figure 8B and/or Figure 8C and/or or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. The program (REP) procedure, and shown in FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) is The procedure of Step D) was shortened to 1 to 8 days, as discussed in detail below and in the Examples and Figures. In some embodiments, the initial first amplification (including a procedure referred to herein as Pre-Rapid Amplification (Pre-REP), and Figure 8 (in particular, such as Figure 8A and/or Figure 8B and/or Figure 8C and/or or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) shown as step B) is shortened to 1 to 7 days, and rapid second amplification (including referred to herein as rapid amplification The program (REP) procedure, and shown in FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) is The procedure of Step D) was shortened to 1 to 9 days, as discussed in detail below and in the Examples and Figures. In some embodiments, the initial first amplification, including a procedure referred to herein as Pre-Rapid Amplification (Pre-REP), and shown in FIG. procedure) is 1 to 7 days, and a rapid second amplification (including a procedure referred to herein as Rapid Amplification Protocol (REP), and Figure 8 (in particular such as Figure 8A and/or Figure 8B and/or Figure 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) shown as step D) is 1 to 10 days, as discussed in detail below and in the Examples and Figures. In some embodiments, the initial first amplification (such as in FIG. 8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8B and/or FIG. 8C and/or FIG. Figure 8F and/or Figure 8G) described as the amplification of step B) was shortened to 8 days, and the rapid second amplification (such as in Figure 8 (especially such as Figure 8A and/or Figure 8B and/or Figure 8C and /or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) described in step D) of amplification) is 7 to 9 days. In some embodiments, the initial first amplification (such as in FIG. 8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8B and/or FIG. 8C and/or FIG. Figure 8F and/or Figure 8G) is described as the amplification of step B) for 8 days, and the rapid second amplification (such as in Figure 8 (especially such as Figure 8A and/or Figure 8B and/or Figure 8C and/or Or the amplification described in step D in FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) is 8 to 9 days. In some embodiments, the initial first amplification (such as in FIG. 8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8B and/or FIG. 8C and/or FIG. Figure 8F and/or Figure 8G) described as the amplification of step B) was shortened to 7 days, and the rapid second amplification (such as in Figure 8 (especially such as Figure 8A and/or Figure 8B and/or Figure 8C and /or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) described in step D) of the amplification) is 7 to 8 days. In some embodiments, the initial first amplification (such as in FIG. 8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8B and/or FIG. 8C and/or FIG. Figure 8F and/or Figure 8G) described as the amplification of step B) was shortened to 8 days, and the rapid second amplification (such as in Figure 8 (especially such as Figure 8A and/or Figure 8B and/or Figure 8C and /or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) described in step D) of amplification) for 8 days. In some embodiments, the initial first amplification (such as in FIG. 8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8B and/or FIG. 8C and/or FIG. Figure 8F and/or Figure 8G) is described as the amplification of step B) for 8 days, and the rapid second amplification (such as in Figure 8 (especially such as Figure 8A and/or Figure 8B and/or Figure 8C and/or Or the amplification described in step D in FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) is 9 days. In some embodiments, the initial first amplification (such as in FIG. 8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8B and/or FIG. 8C and/or FIG. Figure 8F and/or Figure 8G) is described as the amplification of step B) for 8 days, and the rapid second amplification (such as in Figure 8 (especially such as Figure 8A and/or Figure 8B and/or Figure 8C and/or Or the amplification described in step D) in Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) is 10 days. In some embodiments, the initial first amplification (such as in FIG. 8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8B and/or FIG. 8C and/or FIG. Figure 8F and/or Figure 8G) is described as the amplification of step B) for 7 days, and the rapid second amplification (such as in Figure 8 (especially such as Figure 8A and/or Figure 8B and/or Figure 8C and/or Or the amplification described in step D in FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) is 7 to 10 days. In some embodiments, the initial first amplification (such as in FIG. 8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8B and/or FIG. 8C and/or FIG. Figure 8F and/or Figure 8G) is described as the amplification of step B) for 7 days, and the rapid second amplification (such as in Figure 8 (especially such as Figure 8A and/or Figure 8B and/or Figure 8C and/or Or the amplification described in step D in FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) is 8 to 10 days. In some embodiments, the initial first amplification (such as in FIG. 8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8B and/or FIG. 8C and/or FIG. Figure 8F and/or Figure 8G) is described as the amplification of step B) for 7 days, and the rapid second amplification (such as in Figure 8 (especially such as Figure 8A and/or Figure 8B and/or Figure 8C and/or Or the amplification described in step D in FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) is 9 to 10 days. In some embodiments, the initial first amplification (such as in FIG. 8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8B and/or FIG. 8C and/or FIG. Figure 8F and/or Figure 8G) described as the amplification of step B) was shortened to 7 days, and the rapid second amplification (such as in Figure 8 (especially such as Figure 8A and/or Figure 8B and/or Figure 8C and /or Figure 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) described in step D) of amplification) is 7 to 9 days. In some embodiments, the combination of an initial first amplification and a rapid second amplification (such as the amplifications described as step B and step D in FIG. 8 (especially, for example, FIG. 1B and/or FIG. 8C )) is 14 - 16 days, as discussed in detail below and in the Examples and Figures. In particular, it is believed that certain embodiments of the invention comprise an initial first expansion step in which TILs are detected by exposure to an anti-CD3 antibody (e.g. OKT-3) in the presence of IL-2 or in the presence of at least IL-2 and anti-CD3 Activation by exposure to antigen in the presence of antibodies (eg OKT-3). In certain embodiments, the TILs activated in the initial first expansion step as described above are the first TIL population, ie, they are the primary cell population.

The following "step" titles A, B, C, etc. refer to FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8G) and with reference to certain non-limiting embodiments described herein. The sequence of steps below and in FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) is exemplary, And this application and the methods disclosed herein contemplate any combination or order of steps, as well as additional steps, repetitions of steps, and/or omissions of steps. A. Step A : Obtaining Patient Tumor Samples

Generally, TILs are initially obtained from patient tumor samples ("primary TILs") or from circulating lymphocytes, such as peripheral blood lymphocytes, including those with TIL-like features, and are then expanded into larger Large populations for further manipulation as described herein were optionally cryopreserved and assessed for phenotype and metabolic parameters as indicators of TIL health.

Patient tumor samples can be obtained using methods known in the art, typically by surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. In general, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. A tumor sample can also be a liquid tumor, such as a tumor obtained from a hematological malignancy. Solid tumors can be any cancer type, including but not limited to breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, stomach cancer and skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, Bladder cancer, breast cancer, triple negative breast cancer and non-small cell lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, useful TILs are obtained from malignant melanoma tumors, as these tumors have been reported to have particularly high levels of TILs.

Once obtained, tumor samples are typically fragmented using sharps into pieces between 1 mm ³ and about 8 mm ³ , with about 2-3 mm ³ being particularly suitable. TILs were cultured from these fragments using enzymatic tumor lysates. Such tumor lysates can be prepared in enzyme medium (such as Roswell Park Cancer Institute (Roswell Park Memorial Institute; RPMI) 1640 buffer, 2 mM glutamic acid, 10 mcg/mL gentamycin (gentamicine), 30 units/mL DNase and 1.0 mg/mL collagenase), followed by mechanical dissociation (eg, using a tissue dissociator). Tumor lysates can be generated by placing tumors in enzymatic medium and mechanically dissociating tumors for approximately 1 minute, followed by incubation at 37°C in 5% _CO for 30 minutes, followed by repeating mechanical dissociation and incubation under the aforementioned conditions Cycle until only small pieces of tissue remain. At the end of this process, if the cell suspension contains large numbers of red blood cells or dead cells, density gradient separation using FICOLL branched-chain hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in US Patent Application Publication No. 2012/0244133 Al, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in a method of expanding TILs or a method of treating cancer in any of the embodiments described herein.

As noted above, in some embodiments, TILs are derived from solid tumors. In some embodiments, solid tumors are not fragmented. In some embodiments, solid tumors are not fragmented and whole tumors are enzymatically lysed. In some embodiments, tumors are lysed in an enzyme mixture comprising collagenase, DNase, and hyaluronidase. In some embodiments, tumors are lysed in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1 to 2 hours. In some embodiments, tumors are lysed in an enzyme mixture comprising collagenase, DNase, and hyaluronidase at 37° C., 5% CO ₂ for 1 to 2 hours. In some embodiments, tumors are lysed in an enzyme mixture comprising collagenase, DNase, and hyaluronidase at 37° C., 5% CO ₂ , with rotation, for 1 to 2 hours. In some embodiments, tumor lines were lysed overnight with constant rotation. In some embodiments, tumors are lysed overnight at 37°C, 5% _CO2 , with constant rotation. In some embodiments, whole tumors are combined with enzymes to form a tumor lysis reaction mixture.

In some embodiments, tumors are reconstituted with lyophilized enzymes in sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of collagenase is a 100 mg/mL 10× working stock solution.

In some embodiments, the enzyme mixture comprises DNase. In some embodiments, the working stock solution of DNase is 10,000 IU/mL 10X working stock solution.

In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is a 10 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase, 1000 IU/mL DNase, and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

Generally, a cell suspension obtained from a tumor is referred to as a "primary cell population" or a "freshly obtained" or "freshly isolated" cell population. In certain embodiments, a freshly obtained population of TIL cells is exposed to a cell culture medium comprising antigen presenting cells, IL-12, and OKT-3.

In some embodiments, fragmentation includes physical fragmentation, including, for example, segmentation and dissolution. In some embodiments, segments are physical segments. In some embodiments, a segment is a segmentation. In some embodiments, the fragments are by dissolution. In some embodiments, TILs can be cultured initially from enzymatic tumor lysates and tumor fragments obtained from patients. In some embodiments, TILs can be cultured initially from enzymatic tumor lysates and tumor fragments obtained from patients.

In some embodiments, when the tumor is a solid tumor, for example, in step A (as shown in FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. The order of steps in Figure 8F and/or Figure 8G) is exemplary, and the present application and the methods disclosed herein contemplate any combination or order of steps, as well as the additional step) provided in ) after obtaining a tumor sample, for Tumors undergo physical fragmentation. In some embodiments, fragmentation occurs prior to cryopreservation. In some embodiments, fragmentation occurs following cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the fragmenting step is an in vitro or ex vivo process. In some embodiments, tumor fragments and 10, 20, 30, 40 or more fragments or pieces are placed in each container for an initial first expansion. In some embodiments, tumors are fragmented and 30 or 40 fragments or blocks are placed in each vessel for an initial first expansion. In some embodiments, tumors are fragmented and 40 fragments or blocks are placed in each vessel for an initial first expansion. In some embodiments, the plurality of fragments comprises from about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm ³ . In some embodiments, the plurality of segments comprises about 30 to about 60 segments having a total volume of about 1300 mm ³ to about 1500 mm ³ . In some embodiments, the plurality of segments comprises about 50 segments having a total volume of about 1350 mm ³ . In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

In some embodiments, TILs are obtained from tumor fragments. In some embodiments, tumor fragments are obtained by sharps segmentation. In some embodiments, the tumor fragment is between about 1 mm ³ and 10 mm ³ . In some embodiments, the tumor fragment is between about 1 mm ³ and 8 mm ³ . In some embodiments, the tumor fragment is about 1 mm ³ . In some embodiments, the tumor fragment is about 2 mm ³ . In some embodiments, the tumor fragment is about 3 mm ³ . In some embodiments, the tumor fragment is about 4 mm ³ . In some embodiments, the tumor fragment is about 5 mm ³ . In some embodiments, the tumor fragment is about 6 mm ³ . In some embodiments, the tumor fragment is about 7 mm ³ . In some embodiments, the tumor fragment is about 8 mm ³ . In some embodiments, the tumor fragment is about 9 mm ³ . In some embodiments, the tumor fragment is about 10 mm ³ . In some embodiments, the tumor fragment is 1 to 4 mm x 1 to 4 mm x 1 to 4 mm. In some embodiments, tumor fragments are 1 mm x 1 mm x 1 mm. In some embodiments, the tumor fragment is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor fragment is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor segment is 4 mm x 4 mm x 4 mm.

In some embodiments, tumors are sectioned to minimize the amount of hemorrhagic, necrotic and/or adipose tissue on each piece. In some embodiments, tumors are sectioned to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, tumors are sectioned to minimize the amount of necrotic tissue on each piece. In some embodiments, tumors are sectioned to minimize the amount of adipose tissue on each piece. In certain embodiments, the tumor fragmentation step is an in vitro or ex vivo method.

In some embodiments, tumor fragmentation is performed so as to maintain tumor internal structure. In some embodiments, tumor fragmentation is performed without a sawing action using a scalpel. In some embodiments, TILs are obtained from tumor lysates. In some embodiments, by incubating in an enzyme medium (such as but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamycin, 30 U/mL DNase, and 1.0 mg/mL collagenase), followed by Mechanical dissociation (GentleMACS, Miltenyi Biotechnology, Auburn, CA) was performed to generate tumor lysates. Tumors can be dissociated mechanically for about 1 minute after placing them in the enzymatic medium. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically disrupted again for about 1 minute. Tumors were mechanically disrupted a third time for approximately 1 min after an additional 30 min incubation at 37°C in 5% _CO2 . In some embodiments, if large pieces of tissue remain after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to _the sample, with or without further incubation at 37°C in 5% CO for 30 minutes. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed to remove these cells.

In some embodiments, the cell suspension prior to the initial first expansion step is referred to as a "primary cell population" or a "freshly obtained" or "freshly isolated" cell population.

In some embodiments, the cells are optionally frozen after isolation of the sample (e.g., after obtaining the tumor sample and/or after obtaining a cell suspension from the tumor sample) and stored frozen prior to proceeding to expansion as described in Step B, This step B is described in further detail below and is illustrated in Fig. 8, in particular eg Fig. 8B. 1. Thick needle/small biopsy-derived TIL

In some embodiments, TILs are initially obtained from patient tumor samples obtained by core biopsy or similar procedures ("primary TILs") and subsequently expanded into larger populations for further manipulation as described herein, Cryopreserved as appropriate and phenotypic and metabolic parameters assessed as appropriate.

In some embodiments, patient tumor samples can be obtained using methods known in the art, typically via small biopsies, coarse needle biopsies, needle biopsies, or other methods used to obtain a mixture containing tumor and TIL cells obtained by means of samples. In general, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. A tumor sample can also be a liquid tumor, such as a tumor obtained from a hematological malignancy. In some embodiments, samples may be from multiple small tumor samples or biopsies. In some embodiments, a sample may comprise multiple tumor samples from a single tumor of the same patient. In some embodiments, a sample may comprise multiple tumor samples from one, two, three or four tumors of the same patient. In some embodiments, a sample may comprise multiple tumor samples from multiple tumors of the same patient. The solid tumor can be lung cancer and/or non-small cell lung cancer (NSCLC).

In general, cell suspensions obtained from tumor cores or fragments are referred to as "primary cell populations" or "freshly obtained" or "freshly isolated" cell populations. In certain embodiments, a freshly obtained population of TIL cells is exposed to a cell culture medium comprising antigen presenting cells, IL-2, and OKT-3.

In some embodiments, removal of a metastatic lesion may be required if the tumor is metastatic and the primary lesion has been effectively treated/removed in the past. In some embodiments, the minimally invasive approach is to remove skin lesions or lymph nodes on the neck or armpit area, if available. In some embodiments, a skin lesion is removed or a small biopsy thereof is removed. In some embodiments, lymph nodes or small biopsies thereof are removed. In some embodiments, the tumor is melanoma. In some embodiments, a small biopsy of melanoma comprises a nevus or a portion thereof.

In some embodiments, the small biopsy is a punch biopsy. In some embodiments, punch biopsies are obtained by pressing a circular blade into the skin. In some embodiments, a punch biopsy is obtained with a circular blade pressed into the skin surrounding the suspected mole. In some embodiments, a punch biopsy is obtained with a circular blade pressed into the skin, and a circular piece of skin is removed. In some embodiments, the small biopsy is a punch biopsy and a circular portion of the tumor is removed.

In some embodiments, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy and the entire mole or growth is removed. In some embodiments, the small biopsy is an excisional biopsy and the entire mole or growth is removed along with a small margin of normal looking skin.

In some embodiments, the small biopsy is an open biopsy. In some embodiments, the small biopsy is an open biopsy and only the most irregular portion of the mole or growth is taken. In some embodiments, the small biopsy is an open biopsy, and the open biopsy is used when other techniques cannot do it, such as when a suspicious mole is very large.

In some embodiments, the small biopsy is a lung biopsy. In some embodiments, a small biopsy is obtained by bronchoscopy. Generally, bronchoscopy involves passing a small tool under the patient's anesthesia through the nose or mouth, down the throat and into the bronchial passages, where the small tool is used to remove some tissue. In some embodiments, where a tumor or growth cannot be reached via bronchoscopy, a transbronchial biopsy may be employed. Generally, for a transbronchial biopsy, the patient is also under anesthesia and a needle is inserted through the skin directly into the suspicious site to remove a small sample of tissue. In some embodiments, transthoracic biopsy may require interventional radiography (eg, using an x-ray or CT scan to guide the needle). In some embodiments, the small biopsy is obtained by needle biopsy. In some embodiments, a small biopsy is obtained endoscopically with ultrasound (eg, endoscope with light attached and placed orally in the esophagus). In some embodiments, the small biopsy is obtained surgically.

In some embodiments, the small biopsy is a head and neck biopsy. In some embodiments, the small biopsy is an open biopsy. In some embodiments, the small biopsy is an open biopsy in which a small piece of tissue is excised from an abnormal-looking area. In some embodiments, samples can be collected without hospitalization if the abnormal area is easily accessible. In some embodiments, if the tumor is deep inside the mouth or throat, the biopsy may need to be performed in an operating room under general anesthesia. In some embodiments, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy, in which an entire area is removed. In some embodiments, the small biopsy is a fine needle aspiration (FNA). In some embodiments, the small biopsy is fine needle aspiration (FNA), in which cells are drawn (aspirated) from the tumor or mass using a very thin needle attached to a syringe. In some embodiments, the small biopsy is a punch biopsy. In some embodiments, the small biopsy is a punch biopsy in which a piece of the suspicious area is removed using punch forceps.

In some embodiments, the small biopsy is a cervical biopsy. In some embodiments, a small biopsy is obtained via a colposcope. Typically, the colposcopy approach employs a lighted magnifying instrument (colposcope) attached to a binocular magnifying glass, which is then used to biopsy a small portion of the cervix. In some embodiments, the small biopsy is a cervical conization/cone biopsy. In some embodiments, the small biopsy is a cervical conization/cone biopsy, where an outpatient procedure may be required to remove larger pieces of tissue from the cervix. In some embodiments, in addition to aiding in the diagnosis, the cone biopsy may also be used as an initial treatment.

The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or areas of fluid. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to malignant, neoplastic or cancerous solid tumors. Solid tumor cancers include lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). The histological structure of solid tumors consists of interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells in which cancer cells are dispersed and which provide a supportive microenvironment.

In some embodiments, a sample from a tumor is obtained as a fine needle aspirate (FNA), a coarse needle biopsy, a small biopsy (including, for example, a punch biopsy). In some embodiments, the sample is placed in the G-Rex 10 first. In some embodiments, when there are 1 or 2 coarse needle biopsy and/or small biopsy samples, the samples are first placed in the G-REX-10. In some embodiments, when there are 3, 4, 5, 6, 8, 9, or 10 or more coarse needle biopsy and/or small biopsy samples, the samples are first Placed in G-REX-100. In some embodiments, when there are 3, 4, 5, 6, 8, 9, or 10 or more coarse needle biopsy and/or small biopsy samples, the samples are first Placed in G-REX-500.

FNA can be obtained from skin tumors including, for example, melanoma. In some embodiments, the FNA is obtained from a skin tumor, such as a skin tumor from a patient with metastatic melanoma. In some instances, the melanoma patient has previously been treated surgically.

FNA can be obtained from lung tumors including, for example, NSCLC. In some embodiments, the FNA is obtained from a lung tumor, such as a lung tumor from a non-small cell lung cancer (NSCLC) patient. In some instances, NSCLC patients have previously been treated surgically.

The TILs described herein can be obtained from FNA samples. In some instances, FNA samples were obtained or isolated from patients using fine-gauge needles in the range of 18-gauge needles to 25-gauge needles. Fine gauge needles can be 18, 19, 20, 21, 22, 23, 24 or 25. In some embodiments, a FNA sample from a patient may contain at least 400,000 TILs, such as 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs , 800,000 TIL, 850,000 TIL, 900,000 TIL, 950,000 TIL or more.

In some instances, the TILs described herein are obtained from crude needle biopsy samples. In some instances, a coarse needle biopsy sample is obtained or isolated from a patient using a surgical or medical needle in the range of 11 gauge to 16 gauge needles. The needles can be 11 gauge, 12 gauge, 13 gauge, 14 gauge, 15 gauge or 16 gauge. In some embodiments, a crude needle biopsy sample from a patient may contain at least 400,000 TILs, such as 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs , 750,000 TIL, 800,000 TIL, 850,000 TIL, 900,000 TIL, 950,000 TIL or more.

Generally, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

In some embodiments, TILs are not obtained from tumor lysates. In some embodiments, the solid tumor core is not fragmented.

In some embodiments, TILs are obtained from tumor lysates. In some embodiments, by incubating in an enzyme medium (such as but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamycin, 30 U/mL DNase, and 1.0 mg/mL collagenase), followed by Mechanical dissociation (GentleMACS, Miltenyi Biotechnology, Auburn, CA) was performed to generate tumor lysates. Tumors can be dissociated mechanically for about 1 minute after placing them in the enzymatic medium. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes, and then mechanically disrupted again for about 1 minute. Tumors were mechanically disrupted a third time for approximately 1 min after an additional 30 min incubation at 37°C in 5% _CO2 . In some embodiments, if large pieces of tissue remain after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to _the sample, with or without further incubation at 37°C in 5% CO for 30 minutes. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed to remove these cells.

In some embodiments, obtaining the first population of TILs comprises a multifocal sampling method.

The tumor dissociation enzyme mix may include one or more dissociation (digestion) enzymes such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, dispase (dispase), Chymotrypsin, chymopapain, trypsin, casein, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociative or Proteolytic enzymes, and any combination thereof.

In some embodiments, a resolvase is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in an amount of sterile buffer, such as Hank's balance salt solution (HBSS).

In some cases, collagenase (such as animal-derived-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The concentration of the freeze-dried stock enzyme can be 2892 PZ U per vial. In some embodiments, the collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, such as from about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL-about 350 PZ U/mL, about 100 PZ U/mL-about 300 PZ U/mL, about 150 PZ U/mL-about 400 PZ U/mL, about 100 PZ U/mL, about 150 PZ U/mL U/mL, about 200 PZ U/mL, about 210 PZ U/mL, about 220 PZ U/mL, about 230 PZ U/mL, about 240 PZ U/mL, about 250 PZ U/mL, about 260 PZ U /mL, about 270 PZ U/mL, about 280 PZ U/mL, about 289.2 PZ U/mL, about 300 PZ U/mL, about 350 PZ U/mL, or about 400 PZ U/mL.

In some embodiments, dispase is reconstituted in 1 ml sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after reconstitution, the neutral protease stock solution ranges from about 100 DMC/mL to about 400 DMC/mL, such as from about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL - about 350 DMC/mL, about 100 DMC/mL - about 300 DMC/mL, about 150 DMC/mL - about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC /mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL, or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 ml sterile HBSS or another buffer. The concentration of lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, such as about 1 KU/mL, about 2 KU/mL, about 3 KU/mL mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, enzyme stock solutions can vary, so the concentration of the lyophilized stock solution is verified and the final amount of enzyme added to the digest mix is modified accordingly.

In some embodiments, the enzyme mix includes about 10.2 μl of dispase (0.36 DMC U/mL), 21.3 μl of collagenase (1.2 PZ/mL), and 250 μl of deoxyribonuclease I in about 4.7 mL of sterile HBSS (200 U/mL). 2. T cells and TIL in pleural effusion

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cells or TILs for expansion according to the procedures described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of T cells or TILs for expansion according to the procedures described herein is a pleural effusion-derived sample. See, eg, the methods described in US Patent Publication US 2014/0295426, which is hereby incorporated by reference in its entirety for all purposes.

In some embodiments, any pleural fluid or pleural effusion suspected of and/or containing TIL may be used. Such samples may be derived from primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, the sample may be secondary metastatic cancer cells originating from another organ, such as breast, ovary, colon, or prostate. In some embodiments, the sample used in the amplification methods described herein is pleural exudate. In some embodiments, the sample used in the amplification methods described herein is a pleural transudate. Other biological samples may include other serum fluids containing TILs, including, for example, ascitic fluid or pancreatic cyst fluid from the abdomen. Ascites and pleural fluids involve very similar chemical systems; both the abdomen and the lungs have mesothelial cell lines and fluid forms in the thoracic and peritoneal cavities in the same malignancy event, and in some embodiments, such fluids contain TIL. In some embodiments of the present invention exemplifying pleural fluid, the same method can be performed using ascites or other cystic fluid containing TIL to obtain similar results.

In some embodiments, pleural fluid is removed directly from the patient in an unprocessed form. In some embodiments, prior to the contacting step, untreated pleural fluid is placed in standard blood collection tubes, such as EDTA or heparin tubes. In some embodiments, prior to the contacting step, untreated pleural fluid was placed in standard CellSave® tubes (Veridex). In some embodiments, samples are placed in CellSave tubes immediately after collection from the patient to avoid a reduction in the number of viable TILs. If retained in untreated pleural fluid, the number of viable TILs may decrease significantly within 24 hours, even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient. In some embodiments, samples are placed in appropriate collection tubes within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

In some embodiments, samples of pleural fluid from selected individuals can be diluted. In some embodiments, the dilution is 1:10 pleural fluid to diluent. In other embodiments, the dilution is 1:9 pleural fluid to diluent. In other embodiments, the dilution is 1:8 pleural fluid to diluent. In other embodiments, the dilution is 1:5 pleural fluid to diluent. In other embodiments, the dilution is 1:2 pleural fluid to diluent. In other embodiments, the dilution is 1:1 pleural fluid to diluent. In some embodiments, the diluent includes saline, phosphate buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, samples were placed in CellSave tubes immediately after collection and dilution from the patient to avoid reduction of viable TILs, which may be present at 4°C if retained in untreated pleural fluid. Visible reduction within 24 to 48 hours. In some embodiments, the pleural fluid sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal from the patient and dilution. In some embodiments, the pleural fluid sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal from the patient and dilution at 4°C.

In another embodiment, the pleural fluid sample is concentrated by conventional means prior to further processing steps. In some embodiments, preconditioning of pleural fluid is preferred in cases where it must be stored frozen for transport to the laboratory performing the method or for subsequent analysis (e.g., after 24 to 48 hours after collection) . In some embodiments, the pleural fluid sample is prepared by centrifuging the pleural fluid sample after it is removed from the individual and resuspending the centrifugate or pellet in buffer. In some embodiments, pleural fluid samples are centrifuged and resuspended multiple times before being frozen for shipping or later analysis and/or processing.

In some embodiments, the pleural fluid sample is concentrated by using filtration methods prior to further processing steps. In some embodiments, the pleural fluid sample used in the contacting step is prepared by filtering the fluid through a filter having a known and substantially uniform pore size that allows pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the pores in the membrane may be at least 4 μM in diameter. In other embodiments, the pore size can be 5 μM or larger, and in other embodiments, can be any of 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. Following filtration, cells retained by the membrane, including TIL, can be washed from the membrane into a suitable physiologically acceptable buffer. Cells (including TILs) concentrated in this way can then be used in the contacting step of the method.

In some embodiments, a pleural fluid sample (including, for example, unprocessed pleural fluid), diluted pleural fluid, or a resuspended cell pellet is contacted with a lysing agent that differentially lyses cells present in the sample. Anucleated red blood cells. In some embodiments, this step is performed prior to further processing steps where the pleural fluid contains a large number of RBCs. Suitable lysis reagents include a single lysis reagent or a lysis reagent and a quenching reagent, or a lysis reagent, a quencher and a fixation reagent. Suitable dissolution systems are commercially available and include the BD Pharm Lyse™ system (Becton Dickenson). Other dissolution systems include the Versalyse™ system, the FACSlyse™ system (BD Medical), the Immunoprep™ system, or the Erythrolyse II system (Beckman Coulter, Inc.) or the ammonium chloride system. In some embodiments, the lysing agent may vary with the prevailing requirements, which are efficient lysis of erythrocytes and conservation of TILs and phenotypic properties of TILs in pleural fluid. In addition to employing a single reagent for lysis, a lysis system suitable for use in the methods described herein may include a second reagent, such as a second reagent that quenches or retards the effect of the lysis reagent during the remaining steps of the method, such as Stabilyse™ Reagents (Beckman Coulter). Depending on the choice of solubilizing reagent or the preferred practice of the method, conventional immobilizing reagents may also be used.

In some embodiments, untreated, diluted, or multiple centrifuged or processed pleural fluid samples as described above are cryopreserved at a temperature of about -140° C., followed by further processing and/or amplification as provided herein . 3. Pre-selection of CD39/CD69 double negative and CD39/CD69 double KO

According to some methods of the invention, TILs are preselected for (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double gene knockout prior to initiating the first amplification, or (iii) A combination of (i) and (ii). In some embodiments, there is also an optional pre-selection step for PD-1.

In some embodiments, a minimum of 3,000 TILs need to be seeded into the initial first expansion. In some embodiments, the pre-selection step yields a minimum of 3,000 TILs. In some embodiments, a minimum of 4,000 TILs need to be seeded into the initial first expansion. In some embodiments, the pre-selection step yields a minimum of 4,000 TILs. In some embodiments, a minimum of 5,000 TILs need to be seeded into the initial first expansion. In some embodiments, the pre-selection step yields a minimum of 5,000 TILs. In some embodiments, a minimum of 6,000 TILs need to be seeded into the initial first expansion. In some embodiments, the pre-selection step yields a minimum of 6,000 TILs. In some embodiments, a minimum of 7,000 TILs need to be seeded into the initial first expansion. In some embodiments, the pre-selection step yields a minimum of 7,000 TILs. In some embodiments, a minimum of 8,000 TILs need to be seeded into the initial first expansion. In some embodiments, the pre-selection step yields a minimum of 8,000 TILs. In some embodiments, a minimum of 9,000 TILs need to be seeded into the initial first expansion. In some embodiments, the pre-selection step yields a minimum of 9,000 TILs. In some embodiments, a minimum of 10,000 TILs need to be seeded into the initial first expansion. In some embodiments, the pre-selection step yields a minimum of 10,000 TILs. In some embodiments, a minimum of 20,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 20,000 TILs. In some embodiments, a minimum of 30,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 30,000 TILs. In some embodiments, a minimum of 40,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 40,000 TILs. In some embodiments, a minimum of 50,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 50,000 TILs. In some embodiments, a minimum of 60,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 60,000 TILs. In some embodiments, a minimum of 70,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 70,000 TILs. In some embodiments, a minimum of 80,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 80,000 TILs. In some embodiments, a minimum of 90,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 90,000 TILs. In some embodiments, a minimum of 100,000 TILs need to be seeded into the first expansion. In some embodiments, the pre-selection step yields a minimum of 100,000 TILs. In some embodiments, the cells are grown or expanded to a density of 200,000. In some embodiments, cells are grown or expanded to a density of 200,000 to provide about 2e8 TILs to initiate a rapid second expansion. In some embodiments, the cells are grown or expanded to a density of 150,000. In some embodiments, cells are grown or expanded to a density of 150,000 to provide about 2e8 TILs to initiate a rapid second expansion. In some embodiments, the cells are grown or expanded to a density of 250,000. In some embodiments, cells are grown or expanded to a density of 250,000 to provide about 2e8 TILs to initiate a rapid second expansion. In some embodiments, the minimum cell density is 10,000 cells to give 10e6 to initiate a rapid second expansion. In some embodiments, the seeding density of 10e6 used to initiate the rapid second expansion can generate more than 1e9 TILs.

In some embodiments, the TIL used to initiate the first amplification is (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) (i ) and (ii) (eg, after preselection and before first amplification). In some embodiments, the TILs used to initiate the first amplification are at least 75% (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) ) a combination of (i) and (ii); at least 80% of (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii)(i) and ( Combination of ii); at least 85% of (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) a combination of (i) and (ii); at least 90% (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double gene knockout, or (iii) a combination of (i) and (ii); at least 95% (i) CD39 /CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) a combination of (i) and (ii); at least 98% of (i) CD39/CD69 double negative and/ or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) a combination of (i) and (ii); or at least 99% (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) a combination of (i) and (ii) (eg, after pre-selection and before initiation of the first amplification).

In some embodiments, pre-diagnosis of CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO is performed by staining primary cell populations, whole tumor digests, and/or whole tumor cell suspension TILs with anti-CD39 and anti-CD69 antibodies. choose. In some embodiments, the anti-CD39 and anti-CD69 antibodies are polyclonal antibodies, such as mouse anti-human CD39 and CD69 polyclonal antibodies, goat anti-human CD39 and CD69 polyclonal antibodies, and the like. In some embodiments, the anti-CD39 and anti-CD69 antibodies are monoclonal antibodies.

In some embodiments, the anti-CD39 antibody used in the preselection is associated with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells expressing CD39 combined. In some embodiments, the anti-CD69 antibody used in the preselection is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% cell binding.

In some embodiments, the patient has been treated with an anti-PD-1 antibody. In some embodiments, the individual has not received anti-PD-1 antibody treatment. In some embodiments, the individual has not been treated with an anti-PD-1 antibody. In some embodiments, the individual has been previously treated with a chemotherapeutic agent. In some embodiments, the individual has previously been treated with a chemotherapeutic agent, but is no longer being treated with that chemotherapeutic agent. In some embodiments, the individual has been treated with a chemotherapeutic agent, or has been treated with an anti-PD-1 antibody. In some embodiments, the individual has been treated with a chemotherapeutic agent and has been treated with an anti-PD-1 antibody. In some embodiments, the patient has not received anti-PD-1 antibody therapy. In some embodiments, the individual is cancer-naïve or treated with a chemotherapeutic agent but not treated with an anti-PD-1 antibody. In some embodiments, the individual is treatment naïve and chemotherapeutic agent treated but not anti-PD-1 antibody treated.

In some embodiments, negative circles are based on FMO. In some embodiments, FACS banking is set after the step of obtaining and/or receiving a first TIL population from a resected tumor of an individual by processing a tumor sample obtained from the individual into tumor fragments. In some embodiments, circles are set for each sort. In some embodiments, a circle is configured for each PBMC sample. In some embodiments, a circle is configured for each PBMC sample. In some embodiments, the circled template is set from PBMCs every 10 days, 20 days, 30 days, 40 days, 50 days, or 60 days. In some embodiments, a circled template is set every 60 days from PBMCs. In some embodiments, the circled template is set for each PBMC sample every 10 days, 20 days, 30 days, 40 days, 50 days, or 60 days. In some embodiments, a circled template is set for each PBMC sample every 60 days.

In some embodiments, the pre-selection involves selecting from the first TIL population (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double gene knockout, or (iii) (i) and The combination of (ii) to obtain a TIL population comprising a selected TIL population from the first TIL population that is at least 11.27% to 74.4% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL. In some embodiments, the first TIL population is at least 10% to 80% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , at least 20% to 80% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 20% to 80% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 30% to 80% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 40% to 80% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 50% to 80% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 10% to 70% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL, at least 20% to 70% CD39/CD69 double negative and/or CD39 ^LO / CD69 ^LO TIL, at least 30% to 70% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL or at least 40% to 70% CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL.

In some embodiments, the selecting step (e.g., pre-selecting and/or selecting for CD39/CD69 double negative cells) comprises the step of: (i) exposing the first TIL population and the PBMC population to an excess of a single CD39- and CD69-binding Strain anti-CD39 IgG and anti-CD69 IgG antibodies, (ii) add excess anti-IgG antibody bound to the fluorophore, (iii) based on the intensity of the fluorophore detected in the PBMC population, in the first CD39/CD69 double negative in the TIL population and/or the intensity of the fluorophore detected in the CD39 ^LO /CD69 ^LO TIL to obtain the CD39/CD69 double negative population, as performed by fluorescence activated cell sorting (FACS) .

In some embodiments, at least 70% of the population of TILs enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs. In some embodiments, at least 80% of the population of TILs enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs. In some embodiments, at least 90% of the population of TILs enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs. In some embodiments, at least 95% of the population of TILs enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs. In some embodiments, at least 99% of the population of TILs enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs. In some embodiments, 100% of the population of TILs enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs.

In some embodiments, the selection step as exemplified in step A3 of process CD39/CD69 GEN 3 in FIG. 8E and/or FIG. 8F and/or FIG. 8G comprises the step of: (i) exposing the first TIL population to an excess Monoclonal anti-CD39 IgG and anti-CD69 IgG antibodies, (ii) adding excess anti-IgG antibodies bound to fluorophores, and (iii) performing fluorophore-based flow cytometry to obtain (i) CD39/ CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) a combination of TIL populations enriched in (i) and (ii).

To determine whether TILs derived from a tumor sample are CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , one skilled in the art can use TILs corresponding to peripheral T cells obtained from blood samples obtained from one or more healthy human individuals. The reference value of CD39 and/or CD69 expression. CD39/CD69 positive cells in a reference sample can be defined using a fluorescence minus one control and a matched isotype control. In some embodiments, immunostaining for CD39/CD69 in tumor-derived TILs is established using the expression of CD39/CD69 measured in CD3+/CD39+/CD69+ peripheral T cells (e.g., reference cells) from healthy individuals Threshold or cut-off value for intensity. The cut-off value can be defined as the maximum intensity of CD39/CD69 immunostaining of CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO T cells. Therefore, TILs with CD39/CD69 expression equal to or below the cut-off value can be considered as CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO cells. In some instances, CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs represent TILs with the lowest intensity of CD39/CD69 immunostaining corresponding to up to 1% or less of total CD3+ cells. In other cases, CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs represent TILs with the lowest intensity of CD39/CD69 immunostaining corresponding to a maximum of 0.75% or less of total CD3+ cells. In some instances, CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs represent TILs with the lowest intensity of CD39/CD69 immunostaining corresponding to a maximum of 0.50% or less of total CD3+ cells. In other cases, CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs represent TILs with the lowest intensity of CD39/CD69 immunostaining corresponding to a maximum of 0.25% or less of total CD3+ cells.

In some embodiments, the protein kinase B (AKT) inhibitor (AKTi) method of Example 16 is used. In some embodiments, the TIL population is cultured in a medium containing an AKT inhibitor to obtain a CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population. In some embodiments, the AKT inhibitor is selected from the group consisting of pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, Oridonin, gossinthin, tenoride, isoliquiritigenin, chrysanthemum, honokiol and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is pataxerti. In some embodiments, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, About 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM or about 5 μM in the culture medium of pataxerti Cultivate TIL populations. b. Fluorophores

In some embodiments, primary cell population TILs are stained with a mixture comprising anti-CD39 and anti-CD69 antibodies linked to a fluorophore and anti-CD3 antibody linked to a fluorophore. In some embodiments, primary cell population TILs are stained with a cocktail comprising anti-CD39 and anti-CD69 antibodies linked to fluorophores (eg, PE, live/dead purple) and anti-CD3-FITC. In some embodiments, primary cell population TILs are stained with a mixture comprising anti-CD39 conjugated to a first fluorophore and anti-CD69 antibody conjugated to a second fluorophore, anti-CD3 conjugated to a third fluorophore Antibodies and live/dead blue dye (such as available from ThermoFisher, MA, Cat. No. L23105), where the primary, secondary, and tertiary antibodies are distinct and capable of individual detection. In some embodiments, following incubation with anti-CD39 and anti-CD69 antibodies, (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) The combination of (i) and (ii) cells is used for amplification according to the first amplification described herein (eg, in Figure 8E and/or Figure 8F and/or in step B of Figure 8G).

In some embodiments, primary cell population TILs are stained with a mixture comprising anti-CD39 and anti-CD69 antibodies linked to a fluorophore and anti-CD3 antibody linked to a fluorophore. In some embodiments, primary cell population TILs are stained with a cocktail comprising anti-CD39 and anti-CD69 antibodies linked to fluorophores (eg, PE, live/dead purple) and anti-CD3-PE-Cy7. In some embodiments, primary cell population TILs are stained with a mixture comprising anti-CD39-FITC and anti-CD69-PE, anti-CD3-PE-Cy7, and live/dead blue dye (ThermoFisher, MA, cat# L23105). In some embodiments, after incubation with anti-CD39 and anti-CD69 antibodies, (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) Combinations of (i) and (ii) cells for use in priming as described herein (e.g., in step B of process CD39/CD69 GEN 3 in Figure 8E and/or Figure 8F and/or Figure 8G ) 1. Amplification of expansion.

In some embodiments, fluorophores include, but are not limited to, PE (Phycoerythrin), APC (Allophycocyanin), PerCP (Dinoxanthin Chlorophyll Protein), DyLight 405, Alexa Fluor 405, Pacific Blue, Alexa Fluor 488, FITC (fluorescent isothiocyanate), DyLight 550, Alexa Fluor 647, DyLight 650 and Alexa Fluor 700. In some embodiments, fluorophores include, but are not limited to, PE-Alexa Fluor® 647, PE-Cy5, PerCP-Cy5.5, PE-Cy5.5, PE-Alexa Fluor® 750, PE-Cy7, and APC -Cy7. In some embodiments, fluorophores include, but are not limited to, luciferin dyes. Examples of luciferin dyes include, but are not limited to, 5-carboxyluciferin, luciferin-5-isothiocyanate, and 6-carboxyluciferin, 5,6-dicarboxyluciferin, 5- (and 6)-sulfoluciferin, pluciferin, succinyl luciferin, 5-(and 6)-carboxy SNARF-1, carboxyluciferin sulfonate, carboxyluciferin zwitterion , Carboxyluciferin quaternary ammonium, carboxyluciferin phosphonate, carboxyluciferin GABA, 5'(6')-carboxyluciferin, carboxyluciferin-cys-Cy5 and luciferin glutathione peptide. In some embodiments, the fluorescent moiety is rose bengal dye. Examples of rose bengal dyes include, but are not limited to, tetramethyl rose bengal-6-isothiocyanate, 5-carboxytetramethyl rose bengal, 5-carboxyrhodol derivatives, carboxy rose bengal 110, Tetramethyl and Tetraethyl Rose Bengal, Diphenyldimethyl and Diphenyldiethyl Rose Bengal, Dinaphthyl Rose Bengal, Rose Bengal 101 Sulfonyl Chloride (sold under the trade name TEXAS RED®). In some embodiments, the fluorescent moiety is a cyanine dye. Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7. 4. Method for expanding peripheral blood lymphocytes (PBL) from peripheral blood

PBL method 1. In some embodiments of the invention, PBLs are amplified using the methods described herein. In some embodiments of the invention, the method comprises obtaining a PBMC sample from whole blood. In some embodiments, the method comprises enriching for T cells by isolating pure T cells from PBMCs using negative selection of the non-CD19+ fraction. In some embodiments, the method comprises enriching T cells by isolating pure T cells from PBMCs using magnetic bead-based negative selection of the non-CD19+ fraction.

In some embodiments of the present invention, PBL method 1 is performed as follows: On day 0, the cryopreserved PBMC sample is thawed and the number of PBMC is counted. T cells were isolated using the Human Pan T Cell Isolation Kit and LS Columns (Miltenyi Biotech).

PBL method 2. In some embodiments of the invention, PBLs are expanded using PBL Method 2, which involves obtaining a PBMC sample from whole blood. T cells from PBMCs were enriched by incubating PBMCs for at least three hours at 37°C and then isolating non-adherent cells.

In some embodiments of the present invention, PBL method 2 is carried out as follows: on day 0, the PMBC sample through cryopreservation is thawed, and the PBMC cells are seeded at 6 million cells per well in CM-2 medium for 6 days. well plate and incubated at 37°C for 3 hours. After 3 hours, non-attached cells (which line the PBL) were removed and their number counted.

PBL method 3. In some embodiments of the invention, PBLs are expanded using PBL Method 3, which involves obtaining a PBMC sample from peripheral blood. B cell lines were isolated using CD19+ selection and T cell lines were selected using negative selection on the non-CD19+ fraction of the PBMC samples.

In some embodiments of the present invention, PBL method 3 is performed as follows: On day 0, cryopreserved PBMCs derived from peripheral blood are thawed and counted. CD19+ B cells were sorted using the CD19 Multi-Sort Human Kit (Miltenyi Biotechnology). In the non-CD19+ cell fraction, T cells were purified using a Human Pan-T Cell Isolation Kit and LS Columns (Miltenyi Biotech).

In some embodiments, PBMCs are isolated from whole blood samples. In some embodiments, PBMC samples are used as starting material for the expansion of PBLs. In some embodiments, samples are cryopreserved prior to the amplification process. In other embodiments, fresh samples are used as starting material for the amplification of PBLs. In some embodiments of the invention, T cells are isolated from PBMCs using methods known in the art. In some embodiments, T cells are isolated using a human pan T cell isolation kit and LS column. In some embodiments of the invention, T cells are isolated from PBMCs using antibody selection methods known in the art (eg CD19 negative selection).

In some embodiments of the invention, the PBMC sample is incubated for a period of time at a temperature effective to identify non-adherent cells. In some embodiments of the invention, the incubation time is about 3 hours. In some embodiments of the invention, the temperature is about 37°C. Non-adherent cells were then expanded using the procedure described above.

In some embodiments, the PBMC sample is from an individual or patient who has been pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor, as appropriate. In some embodiments, the tumor sample is from an individual or patient who has been pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from an individual or patient who has been pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or longer. In other embodiments, PBMCs are derived from patients currently on an ITK inhibitor regimen such as ibrutinib.

In some embodiments, the PBMC sample is from an individual or patient who has been pretreated with a regimen comprising a kinase inhibitor or ITK inhibitor and is refractory to treatment with a kinase inhibitor or ITK inhibitor such as ibrutinib.

In some embodiments, the PBMC sample is from an individual or patient who has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but is no longer on kinase inhibitor or ITK inhibitor treatment. In some embodiments, the PBMC sample is from pre-treatment with a regimen comprising a kinase inhibitor or an ITK inhibitor but no longer on a kinase inhibitor or ITK inhibitor and has not been treated for at least 1 month, at least 2 months months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or longer. In other embodiments, the PBMCs are derived from patients who were previously exposed to an ITK inhibitor but have not been treated for at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

In some embodiments of the invention, at day 0, cells are selected for CD19+ and sorted accordingly. In some embodiments of the invention, antibody-bound beads are used for selection. In some embodiments of the invention, pure T cells are isolated from PBMCs at day 0.

In some embodiments of the invention, for patients not pretreated with ibrutinib or other ITK inhibitors, 10-15 mL of buffy coat will yield about 5 x ¹⁰⁹ PBMCs, which in turn will yield about 5.5 × ¹⁰⁷ PBLs.

In some embodiments of the invention, for patients pretreated with ibrutinib or other ITK inhibitors, the expansion process will yield about 20 x ¹⁰⁹ PBLs. In some embodiments of the invention, 40.3 x ¹⁰⁶ PBMCs will yield about 4.7 x ¹⁰⁵ PBLs.

In any of the foregoing embodiments, PBMCs may be derived from a whole blood sample, obtained by apheresis, derived from buffy coat, or obtained from any other method known in the art for obtaining PBMCs.

In some embodiments, PBLs are prepared using the methods described in US Patent Application Publication No. US 2020/0347350 A1, the disclosure of which is incorporated herein by reference. 5. Method for expanding bone marrow-infiltrating lymphocytes (MIL) from bone marrow-derived PBMCs

MIL method 3. In some embodiments of the invention, the method comprises obtaining PBMCs from bone marrow. On day 0, PBMCs were selected and sorted for CD3+/CD33+/CD20+/CD14+ and the non-CD3+/CD33+/CD20+/CD14+ cell fraction was sonicated and a portion of the sonicated cell fraction was added back to the selected in the cell fraction.

In some embodiments of the present invention, MIL Method 3 is performed as follows: On day 0, a cryopreserved PBMC sample is thawed and the number of PBMCs is counted. Cells were stained with CD3, CD33, CD20 and CD14 antibodies and sorted using a S3e cell sorter (Bio-Rad). Cells were sorted into two fractions: immune cell fraction (MIL fraction) (CD3+CD33+CD20+CD14+) and AML blast cell fraction (non-CD3+CD33+CD20+CD14+).

In some embodiments of the invention, the PBMCs are obtained from bone marrow. In some embodiments, PBMCs are obtained from bone marrow by apheresis, aspiration, biopsy, or other similar means known in the art. In some embodiments, PBMCs are fresh. In other embodiments, the PBMCs are cryopreserved.

In some embodiments of the invention, MIL is expanded from 10-50 mL of bone marrow aspirate. In some embodiments of the invention, 10 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 20 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 30 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 40 mL of bone marrow aspirate is obtained from the patient. In other embodiments, 50 mL of bone marrow aspirate is obtained from the patient.

In some embodiments of the invention, the number of PBMCs obtained from about 10-50 mL of bone marrow aspirate is from about 5×10 ⁷ to about 10×10 ⁷ PBMCs. In other embodiments, the number of PMBCs produced is about 7 x ¹⁰⁷ PBMCs.

In some embodiments of the invention, about 5 x 10 ⁷ to about 10 x 10 ⁷ PBMCs yield about 0.5 x 10 ⁶ to about 1.5 x 10 ⁶ MILs. In some embodiments of the invention, about 1 x ¹⁰⁶ MILs are produced.

In some embodiments of the invention, 12 x ¹⁰⁶ PBMCs derived from bone marrow aspirate yielded approximately 1.4 x ¹⁰⁵ MILs.

In any of the foregoing embodiments, PBMCs may be derived from a whole blood sample, bone marrow, obtained by apheresis, derived from buffy coat, or obtained from any other method known in the art for obtaining PBMCs.

In some embodiments, the MIL is prepared using the methods described in US Patent Application Publication No. US 2020/0347350 A1, the disclosure of which is incorporated herein by reference. B. Step B : Initiate the first amplification

In some embodiments, the methods of the invention provide younger TILs that may provide additional TILs compared to older TILs (i.e., TILs that have further replicated more times prior to administration to an individual/patient). Therapeutic benefit. The characteristics of young TILs have been described in the literature, for example in Donia et al., Scandinavian Journal of Immunology 2012, 75, 157-167; Dudley et al., Clin Cancer Research 2010, 16, 6122- 6131; Huang et al., Journal of Immunotherapy 2005, 28 , 258-267; Besser et al., Journal of Clinical Cancer Research 2013, 19 , OF1-OF9; Besser et al., Journal of Immunotherapy 2009 , 32, 415 -423; Robbins et al., Journal of Immunology 2004 , 173, 7125-7130; Shen et al., Journal of Immunotherapeutics, 2007, 30, 123-129; Zhou et al., Journal of Immunotherapeutics 2005, 28 , 53-62; and Tran et al., Journal of Immunotherapy, 2008 , 31 , 742-751, each of which is incorporated herein by reference.

Following segmentation or digestion of tumor fragments and/or tumor fragments as described, for example, in step A of FIG. 8 (especially, for example, FIG. 8A and/or FIG. Tumor and other cells are grown in serum containing IL-2, OKT-3 and feeder cells (eg, antigen presenting feeder cells). In some embodiments, IL-2, OKT-3, and feeder cells are added together with tumor lysates and/or tumor fragments at the initiation of culture (eg, on day 0). In some embodiments, tumor digests and/or tumor fragments are incubated in containers with up to 60 fragments per container in the presence of 6000 IU/mL IL-2. In some embodiments, this primary cell population is cultured for a period of several days, typically 1 to 8 days, resulting in a bulk TIL population of typically about 1 x ¹⁰⁸ host TIL cells. In some embodiments, this primary cell population is cultured for a period of several days, typically 1 to 7 days, to produce a bulk TIL population, typically about 1 x ¹⁰⁸ bulk TIL cells. In some embodiments, the initial first expansion occurs over a period of 1 to 8 days, resulting in a subject TIL population of typically about 1 x ¹⁰⁸ subject TIL cells. In some embodiments, the initial first expansion occurs over a period of 1 to 7 days, resulting in a subject TIL population of typically about 1 x ¹⁰⁸ subject TIL cells. In some embodiments, this initial first expansion occurs over a period of 5 to 8 days, resulting in a subject TIL population of typically about 1 x ¹⁰⁸ subject TIL cells. In some embodiments, this initial first expansion occurs over a period of 5 to 7 days, resulting in a subject TIL population of typically about 1 x ¹⁰⁸ subject TIL cells. In some embodiments, this initial first expansion occurs over a period of about 6 to 8 days, resulting in a subject TIL population of typically about 1 x ¹⁰⁸ subject TIL cells. In some embodiments, this initial first expansion occurs over a period of about 6 to 7 days, resulting in a subject TIL population of typically about 1 x ¹⁰⁸ subject TIL cells. In some embodiments, this initial first expansion occurs over a period of about 7 to 8 days, resulting in a subject TIL population of typically about 1 x ¹⁰⁸ subject TIL cells. In some embodiments, this initial first expansion occurs over a period of about 7 days, resulting in a subject TIL population of typically about 1 x ¹⁰⁸ subject TIL cells. In some embodiments, this initial first expansion occurs over a period of about 8 days, resulting in a subject TIL population of typically about 1 x ¹⁰⁸ subject TIL cells.

In some embodiments, TILs can be amplified using an initial first amplification step as described below and herein (eg, Figure 8 (in particular, eg, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. The culture initially contained feeder cells; followed by a rapid second expansion as described below in Step D and herein (Step D, including a procedure known as the Rapid Expansion Protocol (REP) step); followed by optional Cryopreserved, and then performed a second step D (including a procedure called the restimulation REP step) as described below and herein. TILs obtained from this process can optionally be characterized for phenotypic characteristics and metabolic parameters as described herein. In some embodiments, the tumor fragment is between about 1 mm ³ and 10 mm ³ .

In some embodiments, the first expansion medium is referred to as "CM" (short for medium). In some embodiments, the CM of Step B consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL Gentamycin.

In some embodiments, there are less than or equal to 240 tumor fragments. In some embodiments, there are less than or equal to 240 tumor fragments placed into less than or equal to 4 containers. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, less than or equal to 60 tumor fragments are placed into 1 container. In some embodiments, each container contains less than or equal to 500 mL of medium per container. In some embodiments, the culture medium comprises IL-2. In some embodiments, the medium comprises 6000 IU/mL IL-2. In some embodiments, the culture medium comprises antigen-presenting feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, the medium comprises 2.5 x ¹⁰⁸ antigen-presenting feeder cells per container. In some embodiments, the medium comprises OKT-3. In some embodiments, the medium comprises 30 ng/mL OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium comprises 6000 IU/mL IL-2, 30 ng OKT-3, and 2.5 x ¹⁰⁸ antigen-presenting feeder cells. In some embodiments, the medium comprises 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 x ¹⁰⁸ antigen presenting feeder cells per container.

Following preparation of tumor fragments, the resulting cells (i.e., fragments of the primary cell population) are cultured in media containing IL-2, antigen-presenting feeder cells, and OKT-3 under conditions favorable for TIL but unfavorable for tumor and other cell growth , and it allows TIL priming and accelerated growth from day 0 culture initiation. In some embodiments, tumor lysates and/or tumor fragments are incubated with 6000 IU/mL IL-2 and antigen presenting feeder cells and OKT-3. This primary cell population is cultured for a period of several days, typically 1 to 8 days, resulting in a bulk TIL population of typically about 1 x ¹⁰⁸ host TIL cells. In some embodiments, the growth medium during initiation of the first expansion comprises IL-2 or a variant thereof and antigen presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured for a period of several days, typically 1 to 7 days, resulting in a bulk TIL population of typically about 1 x ¹⁰⁸ host TIL cells. In some embodiments, the growth medium during initiation of the first expansion comprises IL-2 or a variant thereof and antigen presenting feeder cells and OKT-3. In some embodiments, the IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20 to 30 x ¹⁰⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20 x ¹⁰⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 25 x ¹⁰⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 30 x ¹⁰⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4 to 8×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5 to 7×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6×10 ⁶ IU/mg IL-2. In some embodiments, IL-2 stock solutions are prepared as described in Example C. In some embodiments, the starting first expansion medium comprises about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, About 6000 IU/mL IL-2 or about 5,000 IU/mL IL-2. In some embodiments, the starting first expansion medium comprises about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the starting first expansion medium comprises about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the starting first expansion medium comprises about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the starting first expansion medium comprises about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the starting first expansion cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the starting first expansion cell culture medium further comprises IL-2. In some embodiments, the starting first expansion cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the starting first expansion cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL , about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the starting first expansion cell culture medium comprises 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or about 8000 IU/mL IL-2.

In some embodiments, the starting first expansion medium comprises about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, About 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the starting first expansion medium comprises about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the starting first expansion medium comprises about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the starting first expansion medium comprises about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the starting first expansion medium comprises about 200 IU/mL IL-15. In some embodiments, the starting first expansion cell culture medium comprises about 180 IU/mL IL-15. In some embodiments, the starting first expansion cell culture medium further comprises IL-15. In some embodiments, the starting first expansion cell culture medium comprises about 180 IU/mL IL-15.

In some embodiments, the starting first expansion medium comprises about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, About 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the starting first expansion medium comprises about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the starting first expansion medium comprises about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the starting first expansion medium comprises about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the starting first expansion medium comprises about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the starting first expansion medium comprises about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the starting first expansion medium comprises about 2 IU/mL IL-21. In some embodiments, the starting first expansion cell culture medium comprises about 1 IU/mL IL-21. In some embodiments, the starting first expansion cell culture medium comprises about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the starting first expansion cell culture medium comprises about 1 IU/mL IL-21.

In some embodiments, the starting first expansion cell culture medium comprises an OKT-3 antibody. In some embodiments, the starting first expansion cell culture medium comprises about 30 ng/mL OKT-3 antibody. In some embodiments, the starting first expansion cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL , about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, ~70 ng/mL, ~80 ng/mL, ~90 ng/mL, ~100 ng/mL, ~200 ng/mL, ~500 ng/mL, and ~1 µg/mL OKT-3 antibody . In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises between 15 ng/mL and 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 30 ng/mL OKT-3 antibody. In some embodiments, the OKT-3 antibody is murozumab. See eg Table 1.

In some embodiments, the initial first expansion cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: Urelumab, Utumumab, EU-101, Fusion proteins and fragments, derivatives, variants, biosimilars and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, the initial first expansion cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 at an initial concentration of about 30 ng/mL in addition to one or more TNFRSF agonists antibody, and wherein the one or more TNFRSF agonists comprise a 4-1BB agonist. In some embodiments, in addition to one or more TNFRSF agonists, the starting first expansion cell culture medium further comprises IL-2 at an initial concentration of about 6000 IU/mL and OKT-3 at an initial concentration of about 30 ng/mL antibody, and wherein the one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, the starting first expansion medium is referred to as "CM" (short for medium). In some embodiments, it is referred to as CM1 (Medium 1). In some embodiments, the CM consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL Gentamycin. In some embodiments, CM is CM1 described in the Examples. In some embodiments, initiating the first expansion is performed in the initial cell culture medium or the first cell culture medium. In some embodiments, the starting first expansion medium or initial cell culture medium or first cell culture medium comprises IL-2, OKT-3 and antigen presenting feeder cells (also referred to herein as feeder cells).

In some embodiments, the medium used in the expansion processes disclosed herein is a serum-free medium or a defined medium. In some embodiments, serum-free or defined media comprise basal cell culture media and serum supplements and/or serum substitutes. In some embodiments, serum-free or defined media are used to prevent and/or reduce experimental variation in part due to batch-to-batch variation of serum-containing media.

In some embodiments, serum-free or defined media comprise basal cell culture media and serum supplements and/or serum substitutes. In some embodiments, basal cell culture media include, but are not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T cell expansion Xeno-free medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI Growth Medium, and Ischoff's Modified Dulbecco's Medium.

In some embodiments, serum supplements or serum replacements include, but are not limited to, one or more of: CTS™ OpTmizer T Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagens Protein precursors, one or more antibiotics and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L - Phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine , reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and trace elements containing Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ , Ni ²⁺ , Rb ⁺ , Sn ²⁺ and Zr ⁴⁺ compounds. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some embodiments, CTS™ OpTmizer™ T Cell Immune Cell Serum Replacement is used with conventional growth media including, but not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dahl Burke's medium.

In some embodiments, the total serum replacement concentration (vol %) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, based on the total volume of the serum-free or defined medium. 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In some embodiments, the total serum replacement concentration is about 3% of the total volume of serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of serum-free or defined medium.

In some embodiments, the serum-free or defined medium is a CTS™ OpTmizer™ T cell expansion SFM (Thermo Fisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM Line Combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement, which is mixed with the supplement before use Together. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 µM.

In some embodiments, the defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (Thermo Fisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM Line Combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement, which is mixed with the supplement before use Together. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L - Glutamine. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 6000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 3000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 1000 IU/mL to approximately 8000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 µM.

In some embodiments, serum-free medium or synthetic medium is supplemented with a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM , or 4 mM to about 5 mM glutamine (ie, GlutaMAX®). In some embodiments, the serum-free medium or synthetic medium is supplemented with glutamine (ie, GlutaMAX®) at a concentration of about 2 mM.

In some embodiments, serum-free medium or synthetic medium is supplemented with a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM , or about 65 mM of 2-mercaptoethanol. In some embodiments, the serum-free medium or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the medium is 55 µM.

In some embodiments, defined media as described in International PCT Publication No. WO/1998/030679, incorporated herein by reference, find use in the present invention. In this publication, a serum-free eukaryotic cell culture medium is described. Serum-free eukaryotic cell culture media include basal cell culture media supplemented with serum-free supplements capable of supporting the growth of cells in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises one or more components selected from the group consisting of, or is obtained by combining one or more components selected from the group consisting of: one or more albumins or albumin substitutes one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, One or more trace elements and one or more antibiotics. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin Ferritin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L - Phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine , reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and trace elements containing Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ , Ni ²⁺ , Rb ⁺ , Sn ²⁺ and Zr ⁴⁺ compounds. In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640 , F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dulbecco's Medium.

In some embodiments, it is determined that the concentration of glycine in the medium is in the range of about 5 to 200 mg/L, the concentration of L-histidine is in the range of about 5 to 250 mg/L, and the concentration of L-isoleucine is The concentration of L-methionine is about 5 to 300 mg/L, the concentration of L-methionine is about 5 to 200 mg/L, the concentration of L-phenylalanine is about 5 to 400 mg/L, and the concentration of L-proline is about 1 to 1000 mg/L, the concentration of L-hydroxyproline is about 1 to 45 mg/L, the concentration of L-serine is about 1 to 250 mg/L, and the concentration of L-threonine is about 10 to 500 mg/L, the concentration of L-tryptophan is about 2 to 110 mg/L, the concentration of L-tyrosine is about 3 to 175 mg/L, and the concentration of L-valine is about 5 to 500 mg/L, the concentration of thiamine is about 1 to 20 mg/L, the concentration of reduced glutathione is about 1 to 20 mg/L, and the concentration of L-ascorbic acid-2-phosphate is about 1 to 200 mg /L, the concentration of iron-saturated transferrin is about 1 to 50 mg/L, the concentration of insulin is about 1 to 100 mg/L, the concentration of sodium selenite is about 0.000001 to 0.0001 mg/L, and albumin ( For example, the concentration of AlbuMAX® I) is about 5000 to 50,000 mg/L.

In some embodiments, the non-trace element fraction of the defined medium is present in the concentration ranges listed in the column heading "Concentration Ranges in 1X Medium" in Table 4. In other embodiments, the non-trace element components of the defined medium are present at the final concentrations listed in Table 4 in the column headed "Preferred Embodiments of 1X Medium". In other embodiments, the defined medium is a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trivial fraction ingredients of the type and concentration listed in the column heading "Preferred Embodiments of Supplements" in Table 4.

In some embodiments, the osmolarity of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmolarity is between about 280 and 310 mOsmol. In some embodiments, defined medium is supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. Defined media can be further supplemented with L-glutamine (at a final concentration of approximately 2 mM), one or more antibiotics, non-essential amino acids (NEAA; at a final concentration of approximately 100 μM), 2-mercaptoethanol (at a final concentration of about 100 μM).

In some embodiments, the defined medium described in Smith et al., " Clin. Transl. Immunology ", 4(1), 2015 (doi: 10.1038/cti.2014.31) can be used in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as basal cell culture medium supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

In some embodiments, the cell culture medium in the first and/or second gas permeable container is unfiltered. The use of unfiltered cell culture media simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second gas-permeable container lacks β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure described in step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 1 to 8 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure described in step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 2 to 8 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure described in step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 3 to 8 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure described in step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 4 to 8 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure described in step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 5 to 8 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure described in Step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 6 to 8 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (comprising such as in FIG. The procedure provided in step B of FIG. 8F and/or FIG. 8G ), which may include a procedure sometimes referred to as pre-REP or initial REP), is 7 to 8 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure provided in Step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 8 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure described in step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 1 to 7 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure described in step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 2 to 7 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure described in step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 3 to 7 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure described in step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 4 to 7 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including a procedure such as that described in step B of FIG. 8 (particularly, for example, FIG. 8B and/or FIG. 8C ), which may include what is sometimes referred to as pre-REP or The procedure for initial REP) was 5 to 7 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure described in Step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 6 to 7 days, as discussed in the Examples and Figures. In some embodiments, a first amplification procedure (including, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. /or the procedure provided in Step B of Figure 8G), which may include what is sometimes referred to as pre-REP or initial REP) for 7 days, as discussed in the Examples and Figures.

In some embodiments, initiation of the first TIL expansion can be performed 1 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 1 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 2 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 2 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 3 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 3 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 4 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 4 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 5 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 5 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 6 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 6 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 7 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of the first TIL expansion can be performed 7 days after fragmentation occurs and/or after initiation of the first initial amplification step.

In some embodiments, the initial first expansion of TILs can be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days. In some embodiments, the first TIL expansion can be performed for 1 to 8 days. In some embodiments, the first TIL expansion can be performed for 1 to 7 days. In some embodiments, the first TIL expansion can be performed for 2 days to 8 days. In some embodiments, the first TIL expansion can be performed for 2 days to 7 days. In some embodiments, the first TIL expansion can be performed for 3 days to 8 days. In some embodiments, the first TIL expansion can be performed for 3 days to 7 days. In some embodiments, the first TIL expansion can be performed for 4 to 8 days. In some embodiments, the first TIL expansion can be performed for 4 days to 7 days. In some embodiments, the first TIL expansion can be performed for 5 to 8 days. In some embodiments, the first TIL expansion can be performed for 5 to 7 days. In some embodiments, the first TIL expansion can be performed for 6 to 8 days. In some embodiments, the first TIL expansion can be performed for 6 to 7 days. In some embodiments, the first TIL expansion can be performed for 7 to 8 days. In some embodiments, the first TIL expansion can be performed for 8 days. In some embodiments, the first TIL expansion can be performed for 7 days.

In some embodiments, a combination of IL-2, IL-7, IL-15 and/or IL-21 is employed as the combination during initiation of the first amplification. In some embodiments, during the initiation of the first amplification, for example, according to FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8F and/or FIG. 8G ) and during the Step B procedure described herein, IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof can be included. In some embodiments, a combination of IL-2, IL-15, and IL-21 is employed as the combination during initiation of the first amplification. In some embodiments, in accordance with FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) and as herein IL-2, IL-15 and IL-21 and any combination thereof can be included during the described Step B procedure.

In some embodiments, the first amplification is initiated, for example according to FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. Or step B of FIG. 8G ), which is performed in a closed system bioreactor. In some embodiments, TIL expansion as described herein is performed using a closed system. In some embodiments, bioreactors are employed. In some embodiments, a bioreactor is used as the container. In some embodiments, the bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor employed is G-REX-10. 1. Feeder cells and antigen-presenting cells

In some embodiments, the initiation of the first amplification procedure described herein (for example comprising the following amplifications, such as in Figure 8 (in particular for example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) described in step B of the expansion, and referred to as pre-REP or initial REP expansion) does not require feeder cells at the start of TIL expansion (in Also referred to herein as "antigen presenting cells"), but are added during the initiation of the first expansion. In some embodiments, the initiation of the first amplification procedure described herein (for example comprising the following amplifications, such as in Figure 8 (in particular for example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) described in step B of the expansion, and referred to as pre-REP or initial REP expansion) does not require feeder cells at the start of TIL expansion (in Also referred to herein as "antigen presenting cells"), but added anytime during days 4-8 during the initiation of the first expansion. In some embodiments, the initiation of the first amplification procedure described herein (for example comprising the following amplifications, such as in Figure 8 (in particular for example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) described in step B of the expansion, and referred to as pre-REP or initial REP expansion) does not require feeder cells at the start of TIL expansion (in Also referred to herein as "antigen presenting cells"), but are added anytime during days 4-7 during the initiation of the first expansion. In some embodiments, the initiation of the first amplification procedure described herein (for example comprising the following amplifications, such as in Figure 8 (in particular for example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) described in step B of the expansion, and referred to as pre-REP or initial REP expansion) does not require feeder cells at the start of TIL expansion (in Also referred to herein as "antigen presenting cells"), but added anytime during days 5-8 during the initiation of the first expansion. In some embodiments, the initiation of the first amplification procedure described herein (for example comprising the following amplifications, such as in Figure 8 (in particular for example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) described in step B of the expansion, and referred to as pre-REP or initial REP expansion) does not require feeder cells at the start of TIL expansion (in Also referred to herein as "antigen presenting cells"), but added anytime during days 5-7 during the initiation of the first expansion. In some embodiments, the initiation of the first amplification procedure described herein (for example comprising the following amplifications, such as in Figure 8 (in particular for example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) described in step B of the expansion, and referred to as pre-REP or initial REP expansion) does not require feeder cells at the start of TIL expansion (in Also referred to herein as "antigen presenting cells"), but added anytime during days 6-8 during the initiation of the first expansion. In some embodiments, the initiation of the first amplification procedure described herein (for example comprising the following amplifications, such as in Figure 8 (in particular for example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) described in step B of the expansion, and referred to as pre-REP or initial REP expansion) does not require feeder cells at the start of TIL expansion (in Also referred to herein as "antigen presenting cells"), but are added at any time during days 6-7 during the initiation of the first expansion. In some embodiments, the initiation of the first amplification procedure described herein (for example comprising the following amplifications, such as in Figure 8 (in particular for example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) described in step B of the expansion, and referred to as pre-REP or initial REP expansion) does not require feeder cells at the start of TIL expansion (in Also referred to herein as "antigen presenting cells"), but were added at any time during day 7 or 8 during the initiation of the first expansion. In some embodiments, the initiation of the first amplification procedure described herein (for example comprising the following amplifications, such as in Figure 8 (in particular for example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) described in step B of the expansion, and referred to as pre-REP or initial REP expansion) does not require feeder cells at the start of TIL expansion (in Also referred to herein as "antigen presenting cells"), but were added anytime during day 7 during the initiation of the first expansion. In some embodiments, the initiation of the first amplification procedure described herein (for example comprising the following amplifications, such as in Figure 8 (in particular for example Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) described in step B of the expansion, and referred to as pre-REP or initial REP expansion) does not require feeder cells at the start of TIL expansion (in Also referred to herein as "antigen presenting cells"), but were added anytime during day 8 during the initiation of the first expansion.

In some embodiments, the initiation of the first amplification procedure described herein (e.g., includes amplification such as that described in step B of FIG. or expansion of the initial REP) requires feeder cells (also referred to herein as "antigen presenting cells") at the initiation of TIL expansion and during the initiation of the first expansion. In many embodiments, the feeder cell line is peripheral blood mononuclear cells (PBMC) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, 2.5 x ¹⁰⁸ feeder cells are used during the initiation of the first expansion. In some embodiments, 2.5 x ¹⁰⁸ feeder cells per vessel are used during the initiation of the first expansion. In some embodiments, 2.5 x ¹⁰⁸ feeder cells per GREX-10 are used during the initiation of the first expansion. In some embodiments, 2.5 x ¹⁰⁸ feeder cells per GREX-100 are used during the initiation of the first expansion.

In general, allogeneic PBMCs were not activated by irradiation or heat treatment, and were used in the REP procedure as described in the Examples, which provide an exemplary protocol for assessing the non-replication capacity of irradiated allogeneic PBMCs.

In some embodiments, if the total number of viable cells at day 14 is less than the initial number of viable cells placed in culture at day 0 initiating the first expansion, the PBMC line is considered replication incompetent and acceptable for use as described herein The TIL amplification procedure.

In some embodiments, if the total number of viable cells cultured on day 7 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells placed in culture on day 0 of initiation of the first expansion, it is considered PBMCs are replication incompetent and can be accepted for the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on day 7 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells placed in culture on day 0 of initiation of the first expansion, it is considered PBMCs are replication incompetent and can be accepted for the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 5 to 60 ng/mL OKT3 antibody and 1000 to 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10 to 50 ng/mL OKT3 antibody and 2000 to 5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20 to 40 ng/mL OKT3 antibody and 2000 to 4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25 to 35 ng/mL OKT3 antibody and 2500 to 3500 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng/ml OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen presenting feeder cells in the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175 , about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400 or about 1:500 . In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, a ratio of about 2.5 x ¹⁰⁸ feeder cells to about 100 x ¹⁰⁶ TILs is required to initiate the first expansion procedure described herein. In some embodiments, the initiation of the first expansion procedure described herein requires a ratio of about 2.5 x ¹⁰⁸ feeder cells to about 50 x ¹⁰⁶ TILs. In other embodiments, about 2.5 x ¹⁰⁸ feeder cells to about 25 x ¹⁰⁶ TILs are required to initiate the first expansion described herein. In other embodiments, about 2.5 x ¹⁰⁸ feeder cells are required to initiate the first expansion described herein. In other embodiments, one-fourth, one-third, five-twelfths, or one-half the number of feeder cells used for rapid second expansion is required to initiate the first expansion.

In some embodiments, the medium in the initial first expansion comprises IL-2. In some embodiments, the medium in the initial first expansion comprises 6000 IU/mL IL-2. In some embodiments, the medium initiating the first expansion comprises antigen presenting feeder cells. In some embodiments, the medium in the initial first expansion comprises 2.5 x ¹⁰⁸ antigen presenting feeder cells per vessel. In some embodiments, the medium in the initial first expansion comprises OKT-3. In some embodiments, the medium comprises 30 ng OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium comprises 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 x ¹⁰⁸ antigen-presenting feeder cells. In some embodiments, the medium comprises 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 x ¹⁰⁸ antigen presenting feeder cells per container. In some embodiments, the medium comprises 500 mL of medium per container of 2.5 x ¹⁰⁸ antigen-presenting feeder cells and 15 µg of OKT-3. In some embodiments, the medium comprises 500 mL medium and 15 μg OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium comprises 500 mL medium, 6000 IU/mL IL-2, 30 ng/mL OKT-3, and 2.5 x ¹⁰⁸ antigen-presenting feeder cells. In some embodiments, the medium comprises 500 mL medium, 6000 IU/mL IL-2, 15 µg OKT-3, and 2.5 x ¹⁰⁸ antigen presenting feeder cells per container. In some embodiments, the medium comprises 500 mL of medium per container of 2.5 x ¹⁰⁸ antigen-presenting feeder cells and 15 µg of OKT-3.

In some embodiments, the initiation of the first expansion procedure described herein requires excess feeder cells than TILs during the second expansion. In many embodiments, the feeder cell line is peripheral blood mononuclear cells (PBMC) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen presenting cells (aAPCs) are used instead of PBMCs.

In general, allogeneic PBMCs were inactivated by irradiation or heat treatment and used in the TIL expansion procedures described herein, including the exemplary procedures described in the Figures and Examples.

In some embodiments, artificial antigen presenting cells are used in place of or in combination with PBMCs in initiating the first expansion. 2. Cytokines and other additives

The expansion methods described herein typically use media with high doses of cytokines, particularly IL-2, as known in the art.

Alternatively, it is also possible to initiate the first expansion of TILs using a combination of cytokines, as described in US Patent Application Publication No. US 2017/0107490 A1 using IL-2, IL-15 and IL- 21, the combination of two or more, the disclosure of this case is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, the latter of which in many embodiments has a specific use. The use of a combination of cytokines is particularly advantageous for the generation of lymphocytes, and especially T cells as described therein. See Table 2, for example.

In some embodiments, step B may also include adding OKT-3 antibody or murozumab to the culture medium, as described elsewhere herein. In some embodiments, step B can also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B can also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. In addition, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists, may be used in the medium during step B, Such as thiazolidinedione compounds, as described in US Patent Application Publication No. US 2019/0307796 A1, the disclosure of which is incorporated herein by reference.

In some embodiments, step B may also include adding protein kinase B (AKT) inhibitor (AKTi) to the culture medium. In some embodiments, the TIL population is cultured in a medium containing an AKT inhibitor to obtain a CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population. In some embodiments, the AKT inhibitor is selected from the group consisting of pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, Oridonin, gossinthin, tenoride, isoliquiritigenin, chrysanthemum, honokiol and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is pataxerti. In some embodiments, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, About 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM or about 5 μM in the culture medium of pataxerti Cultivate TIL populations. C. Step C : Transition from Initiating First Amplification to Rapid Second Amplification

In some cases, a subject TIL population obtained from initiating a first amplification (which may include amplification sometimes referred to as pre-REP), including, for example, obtained from, for example, FIG. 8 (in particular, such as FIG. 8A and/or 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. Expansion called Rapid Expansion Protocol (REP)) and then cryopreserved as discussed below. Similarly, where genetically modified TILs are to be used in therapy, either the expanded TIL population from the initial first expansion or the expanded TIL population from the rapid second expansion can be preceded by the expansion step or Genetic modification for appropriate therapy is performed after the initiation of the first expansion and prior to the rapid second expansion.

In some embodiments, obtained from initiating a first amplification (such as in FIG. 8 (in particular such as in FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. / or TILs from step B) indicated in Fig. 8G) were stored until phenotyping for selection. In some embodiments, obtained from initiating a first amplification (such as in FIG. 8 (in particular such as in FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. / or TILs from step B) indicated in Fig. 8G) were not stored and directly subjected to a rapid second amplification. In some embodiments, the TILs obtained from the initiation of the first expansion are not cryopreserved after the initiation of the first expansion and before the rapid second expansion. In some embodiments, the transition from the first amplification to the second amplification is initiated about 2 days, 3 days, 4 days, 5 days, after tumor fragmentation occurs and/or after initiation of the first initial amplification step. Occurs in 6, 7 or 8 days. In some embodiments, the transition from initial first amplification to rapid second amplification occurs about 3 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initial first amplification to rapid second amplification occurs about 3 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs about 4 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs about 4 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs about 5 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs about 5 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs about 6 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs about 6 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs about 7 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs about 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs about 8 days after fragmentation occurs and/or after initiation of the first initial amplification step.

In some embodiments, the transition from initiation of first amplification to rapid second amplification occurs 1 day, 2 days, 3 days, 4 days, 5 days after fragmentation occurs and/or after initiation of the first initial amplification step. Days, 6 days, 7 days or 8 days. In some embodiments, the transition from initial first amplification to rapid second amplification occurs 1 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiation of first amplification to rapid second amplification occurs 1 to 8 days after fragmentation occurs and/or initiation of the first initial amplification step. In some embodiments, the transition from initiation of first amplification to second amplification occurs 2 to 7 days after fragmentation occurs and/or initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs 2 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs between 3 and 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiating the first amplification to the second amplification occurs between 3 and 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initial first amplification to rapid second amplification occurs 4 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initial first amplification to rapid second amplification occurs 4 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initial first amplification to rapid second amplification occurs 5 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initial first amplification to rapid second amplification occurs 5 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initial first amplification to rapid second amplification occurs 6 to 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, The transition from the initiation of the first amplification to the rapid second amplification occurs 6 to 8 days after fragmentation has occurred and/or after initiation of the first initial amplification step. In some embodiments, the transition from initial first amplification to rapid second amplification occurs 7 to 8 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, the transition from initiation of first amplification to rapid second amplification occurs 7 days after fragmentation occurs and/or after initiation of the first initial amplification step. In some embodiments, initiation of first The transition from amplification to rapid second amplification occurs 8 days after fragmentation occurs and/or after initiation of the first initial amplification step.

In some embodiments, the TILs are not stored after initiation of the first amplification and prior to the rapid second amplification, and the TILs are directly subjected to the rapid second amplification (eg, in some embodiments, as shown in FIG. 8( In particular during the transition from step B to step D shown in, for example, Fig. 8A and/or Fig. 8B and/or Fig. 8C and/or Fig. 8D and/or Fig. 8E and/or Fig. 8F and/or Fig. 8G) store). In some embodiments, the transformation occurs in a closed system as described herein. In some embodiments, TILs from the initial first expansion (the second TIL population) are directly subjected to a rapid second expansion without a transition period.

In some embodiments, the transition from first amplification to rapid second amplification is initiated, for example according to FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. Step C of Fig. 8E and/or Fig. 8F and/or Fig. 8G) is performed in a closed system bioreactor. In some embodiments, TIL expansion as described herein is performed using a closed system. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is, for example, GREX-10 or GREX-100. In some embodiments, the closed system bioreactor is a single bioreactor. In some embodiments, the transition from an initial first expansion to a rapid second expansion involves a vertical scale-up of vessel size. In some embodiments, the initial first amplification is performed in a smaller vessel than the rapid second amplification. In some embodiments, the initial first amplification is performed in GREX-100 and the rapid second amplification is performed in GREX-500. D. Step D : Rapid Second Amplification

In some embodiments, after collection and initiation of the first expansion, the TIL cell population is subjected to step A and step B, and the transition referred to as step C (as shown in FIG. and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) followed by further amplification. This further amplification, referred to herein as rapid second amplification or rapid amplification, may include an amplification procedure commonly referred to in the art as a rapid amplification procedure (rapid amplification protocol or REP); and as shown in FIG. 8 (in particular the procedure indicated in step D of eg FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G )). Rapid secondary expansion is typically accomplished in gas-permeable vessels using media containing various components, including feeder cells, sources of cytokines, and anti-CD3 antibodies. In some embodiments, TILs are transferred 1 day, 2 days, 3 days, or 4 days after initiation of rapid second amplification (i.e., on day 8, 9, 10, or 11 of the overall Gen 3 process) into larger volume containers.

In some embodiments, rapid second amplification of TILs (which may include amplification sometimes referred to as REP; and 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G (step D) indicated in step D) can be performed using any TIL culture flask or container known to those skilled in the art. In some embodiments, the second TIL expansion can be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of the rapid second expansion. sky. In some embodiments, the second TIL expansion can be performed about 1 day to about 9 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 1 day to about 10 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 2 days to about 9 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 2 days to about 10 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 3 days to about 9 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 3 days to about 10 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 4 days to about 9 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 4 days to about 10 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 5 days to about 9 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 5 days to about 10 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 6 days to about 9 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 6 days to about 10 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 7 days to about 9 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 7 days to about 10 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 8 days to about 9 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 8 days to about 10 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 9 days to about 10 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 1 day after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 2 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 3 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 4 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 5 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 6 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 7 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 8 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 9 days after initiation of the rapid second expansion. In some embodiments, the second TIL expansion can be performed about 10 days after initiation of the rapid second expansion.

In some embodiments, rapid second amplification can be performed in a gas-permeable container using the methods of the disclosure (including, for example, amplification known as REP; The procedure indicated in step D of FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) is executed. In some embodiments, TILs are expanded in the presence of IL-2, OKT-3, and feeder cells (also referred to herein as "antigen presenting cells") in a rapid second expansion. In some embodiments, TILs are expanded in the presence of IL-2, OKT-3, and feeder cells in a rapid second expansion, wherein the feeder cells are added to the final concentration that was present in the initial first expansion. Two times, 2.4 times, 2.5 times, 3 times, 3.5 times or 4 times the concentration of the feeder cells in the growth. For example, TILs can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulators can include, for example, anti-CD3 antibodies such as about 30 ng/ml OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Raritan, NJ or Auburn, CA Miltenyi Biotechnology, Inc., San Diego, California) or UHCT-1 (available from BioLegend, San Diego, CA, USA). TILs can be amplified to induce further stimulation of TILs in vitro by including one or more cancer antigens (including antigenic portions thereof such as epitopes) during a second expansion, optionally in T cell growth factors ( Expressed from a carrier, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) in the presence of IL-2 or IL-15, such as 300 IU/mL, as appropriate or gpl 00:209-217(210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same cancer antigen pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of the second amplification. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL , about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL- 2. In some embodiments, the cell culture medium comprises 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL , 7000 to 8000 IU/mL, or 8000 IU/mL IL-2.

In some embodiments, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL , about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 µg/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, and 50 ng/mL to 100 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises between 15 ng/mL and 30 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises between 30 ng/mL and 60 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL OKT-3. In some embodiments, the cell culture medium comprises about 60 ng/mL OKT-3. In some embodiments, the OKT-3 antibody is murozumab.

In some embodiments, the medium in the rapid second expansion comprises IL-2. In some embodiments, the medium comprises 6000 IU/mL IL-2. In some embodiments, the medium in the rapid second expansion comprises antigen presenting feeder cells. In some embodiments, the medium in the rapid second expansion comprises 7.5 x ¹⁰⁸ antigen presenting feeder cells per container. In some embodiments, the medium in the rapid second expansion comprises OKT-3. In some embodiments, the medium in the rapid second expansion comprises 500 mL medium and 30 μg OKT-3 per container. In some embodiments, the container is a G-REX-100 MCS flask. In some embodiments, the medium in the rapid second expansion comprises 6000 IU/mL IL-2, 60 ng/mL OKT-3, and 7.5 x ¹⁰⁸ antigen presenting feeder cells. In some embodiments, the medium comprises 500 mL of medium per container with 6000 IU/mL IL-2, 30 µg OKT-3, and 7.5 x ¹⁰⁸ antigen presenting feeder cells.

In some embodiments, the medium in the rapid second expansion comprises IL-2. In some embodiments, the medium comprises 6000 IU/mL IL-2. In some embodiments, the medium in the rapid second expansion comprises antigen presenting feeder cells. In some embodiments, the medium comprises 5 x ¹⁰⁸ to 7.5 x ¹⁰⁸ antigen-presenting feeder cells per vessel. In some embodiments, the medium in the rapid second expansion comprises OKT-3. In some embodiments, the medium in the rapid second expansion comprises 500 mL medium and 30 μg OKT-3 per vessel. In some embodiments, the container is a G-REX-100 MCS flask. In some embodiments, the medium in the rapid second expansion comprises 6000 IU/mL IL-2, 60 ng/mL OKT-3, and 5×10 ⁸ to 7.5×10 ⁸ antigen-presenting feeder cells. In some embodiments, the medium in the rapid second expansion comprises 500 mL medium with 6000 IU/mL IL-2, 30 µg OKT-3, and 5 x ¹⁰⁸ to 7.5 x ¹⁰⁸ antigen-presenting feeder cells per container.

In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: Urelumab, Utumumab, EU-101, Fusion proteins and fragments, derivatives, variants, biosimilars and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 μg/mL to 40 μg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and an OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein the one The or more TNFRSF agonists comprise 4-1BB agonists.

In some embodiments, a combination of IL-2, IL-7, IL-15 and/or IL-21 is employed as the combination during the second expansion. In some embodiments, during the second amplification, including, for example, in accordance with FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8F and/or FIG. 8G ), and during the Step D procedure described herein, IL-2, IL-7, IL-15 and/or IL-21, and any combination thereof, may be included. In some embodiments, a combination of IL-2, IL-15 and IL-21 is employed as the combination during the second expansion. In some embodiments, in accordance with FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) and as herein IL-2, IL-15 and IL-21 and any combination thereof may be included during the described Step D procedure.

In some embodiments, the second expansion can be performed in supplemented cell culture medium comprising IL-2, OKT-3, antigen presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3 and antigen presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen presenting cells (APCs; also known as antigen presenting feeder cells). In some embodiments, the second expansion occurs in cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (ie, antigen-presenting cells).

In some embodiments, the second expansion medium comprises about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 200 IU/mL IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15.

In some embodiments, the second expansion medium comprises about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21 twenty one. In some embodiments, the second expansion medium comprises about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 2 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and/or antigen presenting cells in the rapid expansion and/or the second expansion is about 1 to 10, about 1 to 15, about 1 to 20, about 1 to 25, About 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:75, about 1:100, about 1:125, about 1:150, about 1:175, About 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500 . In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1:100 and 1:200.

In some embodiments, REP and/or rapid second expansion is performed in culture flasks with bulk TIL and a 100-fold or 200-fold excess of deactivated feeder cells, 30 ng/mL OKT3 anti-CD3 antibody, and 6000 IU/mL IL-2 was mixed in 150 ml culture medium, wherein the feeder cell concentration was at least 1.1 times (1.1X), 1.2X, 1.3X, 1.4X, 1.5X, 1.6X, 1.7X, 1.8X, 1.8X, 2X, 2.1X, 2.2X, 2.3X, 2.4X, 2.5X, 2.6X, 2.7X, 2.8X, 2.9X, 3.0X, 3.1X, 3.2X, 3.3X, 3.4X, 3.5X, 3.6X, 3.7X, 3.8X, 3.9X, or 4.0X. The medium was replaced (typically by aspirating 2/3 of the spent medium and replacing it with an equal volume of fresh medium) until the cells were transferred to a replacement growth chamber. Alternative growth chambers include G-REX flasks and gas permeable containers, as discussed more fully below.

In some embodiments, the rapid second expansion (which may include a process known as the REP process) is 7 to 9 days, as discussed in the Examples and Figures. In some embodiments, the second expansion is 7 days. In some embodiments, the second expansion is 8 days. In some embodiments, the second expansion is 9 days.

In some embodiments, the second amplification (which may include the amplification referred to as REP, and in FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. Figure 8E and/or Figure 8F and/or Figure 8G) The amplification mentioned in Step D) can be prepared in a gas-permeable culture flask (G-REX-100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA), 5×106 or 10×106 TILs can be mixed with PBMCs in 400 mL supplemented with 5% human AB serum, 3000 IU/mL IL-2, and Cultured in 30 ng/ml anti-CD3 (OKT3) 50/50 medium. G-REX-100 flasks can be incubated at 37°C in 5% CO ₂ . On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 xg) for 10 minutes. The TIL pellet can be resuspended with 150 mL of fresh medium containing 5% human AB serum, 6000 IU/mL IL-2, and added back to the original GREX-100 culture flask. When TILs are serially expanded in GREX-100 flasks, at day 10 or 11 the TILs can be moved to a larger flask, such as GREX-500. Cells can be harvested on day 14 of culture. Cells can be harvested on day 15 of culture. Cells can be harvested on day 16 of culture. In some embodiments, the medium is replaced until the cells are transferred to an alternate growth chamber. In some embodiments, 2/3 of the medium is replaced by aspirating spent medium and replacing it with an equal volume of fresh medium. In some embodiments, alternative growth chamber chambers include GREX flasks and gas permeable containers, as discussed more fully below.

In some embodiments, the medium used in the expansion processes disclosed herein is a serum-free medium or a defined medium. In some embodiments, serum-free or defined media comprise basal cell culture media and serum supplements and/or serum substitutes. In some embodiments, serum-free or defined media are used to prevent and/or reduce experimental variation in part due to batch-to-batch variation of serum-containing media.

In some embodiments, serum-free or defined media comprise basal cell culture media and serum supplements and/or serum substitutes. In some embodiments, basal cell culture media include, but are not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T cell expansion Xeno-free medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI Growth Medium, and Ischoff's Modified Dulbecco's Medium.

In some embodiments, serum supplements or serum replacements include, but are not limited to, one or more of: CTS™ OpTmizer T Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagens Protein precursors, one or more antibiotics and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L - Phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine , reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and trace elements containing Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ , Ni ²⁺ , Rb ⁺ , Sn ²⁺ and Zr ⁴⁺ compounds. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some embodiments, CTS™ OpTmizer™ T Cell Immune Cell Serum Replacement is used with conventional growth media including, but not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dahl Burke's medium.

In some embodiments, the total serum replacement concentration (vol %) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, based on the total volume of the serum-free or defined medium. 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In some embodiments, the total serum replacement concentration is about 3% of the total volume of serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of serum-free or defined medium.

In some embodiments, the serum-free or defined medium is a CTS™ OpTmizer™ T cell expansion SFM (Thermo Fisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention.

CTS™ OpTmizer™ T Cell Expansion SFM Line Combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement, which is mixed with the supplement before use Together. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol.

In some embodiments, the defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (Thermo Fisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM Line Combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement, which is mixed with the supplement before use Together. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L - Glutamine. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific), 55 mM 2-Mercaptoethanol, and 2 mM L-Bran Amino acid, and further comprising about 6000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 3000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, the CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific) and about 2 mM Glutamine, and further comprises about 6000 IU/mL IL-2.

In some embodiments, serum-free medium or synthetic medium is supplemented with a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM or 4 mM to about 5 mM glutamine (ie, GlutaMAX®). In some embodiments, the serum-free medium or synthetic medium is supplemented with glutamine (ie, GlutaMAX®) at a concentration of about 2 mM.

In some embodiments, serum-free medium or synthetic medium is supplemented with a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM , or about 65 mM of 2-mercaptoethanol. In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM.

In some embodiments, the synthetic media described in International Patent Application Publication No. WO 1998/030679 and U.S. Patent Application Publication No. US 2002/0076747 A1 , incorporated herein by reference, may be used in the present invention . In this publication, a serum-free eukaryotic cell culture medium is described. Serum-free eukaryotic cell culture media include basal cell culture media supplemented with serum-free supplements capable of supporting the growth of cells in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises one or more components selected from the group consisting of, or is obtained by combining one or more components selected from the group consisting of: one or more albumins or albumin substitutes one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, One or more trace elements and one or more antibiotics. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or β-mercaptoethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin Ferritin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L - Phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine , reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and trace elements containing Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ , Ni ²⁺ , Rb ⁺ , Sn ²⁺ and Zr ⁴⁺ compounds. In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640 , F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dulbecco's Medium.

In some embodiments, it is determined that the concentration of glycine in the medium is in the range of about 5 to 200 mg/L, the concentration of L-histidine is in the range of about 5 to 250 mg/L, and the concentration of L-isoleucine is The concentration of L-methionine is about 5 to 300 mg/L, the concentration of L-methionine is about 5 to 200 mg/L, the concentration of L-phenylalanine is about 5 to 400 mg/L, and the concentration of L-proline is about 1 to 1000 mg/L, the concentration of L-hydroxyproline is about 1 to 45 mg/L, the concentration of L-serine is about 1 to 250 mg/L, and the concentration of L-threonine is about 10 to 500 mg/L, the concentration of L-tryptophan is about 2 to 110 mg/L, the concentration of L-tyrosine is about 3 to 175 mg/L, and the concentration of L-valine is about 5 to 500 mg/L mg/L, the concentration of thiamine is about 1 to 20 mg/L, the concentration of reduced glutathione is about 1 to 20 mg/L, and the concentration of L-ascorbic acid-2-phosphate is about 1 to 200 mg /L, the concentration of iron-saturated transferrin is about 1 to 50 mg/L, the concentration of insulin is about 1 to 100 mg/L, the concentration of sodium selenite is about 0.000001 to 0.0001 mg/L, and albumin ( For example, the concentration of AlbuMAX® I) is about 5000 to 50,000 mg/L.

In some embodiments, the non-trace element fraction components of the defined medium are present in the concentration ranges listed in the column in Table 4 under the heading "Concentration Ranges in 1X Medium". In other embodiments, the final concentrations of the non-trace element fraction components present in the defined medium are listed in Table 4 in the column under the heading "Preferred Embodiments of 1X Medium". In other embodiments, the defined medium is a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trivial fraction ingredients of the type and concentration listed in the column heading "Preferred Embodiments of Supplements" in Table 4.

In some embodiments, the osmolarity of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmolarity is between about 280 and 310 mOsmol. In some embodiments, defined medium is supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. Defined media can be further supplemented with L-glutamine (at a final concentration of approximately 2 mM), one or more antibiotics, non-essential amino acids (NEAA; at a final concentration of approximately 100 μM), 2-mercaptoethanol (at a final concentration of about 100 μM). In some embodiments, defined media as described in Smith et al., Clinical and Translational Immunology, 4(1), 2015 (doi: 10.1038/cti.2014.31 ) can be used in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as basal cell culture medium supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

In some embodiments, the cell culture medium in the first and/or second gas permeable container is unfiltered. The use of unfiltered cell culture media simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second gas-permeable container lacks β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some embodiments, a rapid second amplification (including amplification called REP) is performed and further comprises a step in which TILs with superior tumor reactivity are selected. Any selection method known in the art may be used. For example, the methods described in US Patent Application Publication No. 2016/0010058 A1 , the disclosure of which is incorporated herein by reference, can be used to select TILs with superior tumor reactivity.

Cell viability assays can optionally be followed by rapid secondary amplification, including amplification known as REP amplification, using standard assays known in the art. For example, a trypan blue exclusion assay, which selectively marks dead cells and allows assessment of viability, can be performed on bulk TIL samples. In some embodiments, TIL samples can be counted and assessed for viability using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

The diverse antigen receptors of T and B lymphocytes are generated by somatic recombination of a limited but large number of gene segments. These gene segments: V (variable region), D (diversity region), J (junction region) and C (constant region) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCR). The present invention provides a method for generating TILs that exhibit and increase the diversity of the T cell repertoire. In some embodiments, TILs obtained by the methods of the invention exhibit increased T cell repertoire diversity. In some embodiments, the TILs obtained in the second expansion exhibit increased T cell repertoire diversity. In some embodiments, increasing diversity is increasing immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, present in immunoglobulin heavy chains. In some embodiments, the diversity is present in immunoglobulins, in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and/or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, TCRab (ie, TCRα/β) expression is increased.

In some embodiments, the rapid second expansion medium (eg, sometimes referred to as CM2 or second cell culture medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid second expansion medium (eg, sometimes referred to as CM2 or second cell culture medium) comprises 6000 IU/mL IL-2, 30 μg/flask OKT-3, and 7.5 μg/flask as discussed in more detail below. × 10 ⁸ antigen-presenting feeder cells (APCs). In some embodiments, the rapid second expansion medium (eg, sometimes referred to as CM2 or second cell culture medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid second expansion medium (eg, sometimes referred to as CM2 or second cell culture medium) comprises 6000 IU/mL IL-2, 30 μg/flask OKT-3, and 5 as discussed in more detail below. × 10 ⁸ antigen-presenting feeder cells (APCs).

In some embodiments, the rapid second amplification, for example according to FIG. 8 (especially for example FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. Step D of Fig. 8G) is performed in a closed system bioreactor. In some embodiments, TIL expansion as described herein is performed using a closed system. In some embodiments, bioreactors are employed. In some embodiments, a bioreactor is used as the container. In some embodiments, the bioreactor used is, for example, G-REX-100 or G-REX-500. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-500.

In some embodiments, the step of rapid second expansion is divided into multiple steps to achieve vertical scale-up of culture by: (a) performing a rapid second expansion by culturing the T cells in the small culture for a period of about 3 to 7 days; and then (b) effecting transfer of the T cells in the small culture to a second vessel larger than the first vessel The T cells from the small culture are cultured in the larger culture in a container, such as a G-REX-500-MCS container, for a period of about 4 to 7 days in this second container.

In some embodiments, the step of rapid second expansion is divided into multiple steps to achieve lateral scale-up of culture by: (a) A rapid second expansion is performed by culturing the T cells in the first small-scale culture for a period of about 3 to 7 days; and then (b) transferring and distributing the T cells from the first small-scale culture to at least 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers of the same size as the first container, wherein In each second vessel, the portion of T cells from the first mini-culture transferred to such second vessel was cultured in the second mini-culture for a period of about 4 to 7 days.

In some embodiments, the first small-scale TIL culture is partitioned into a plurality of about 2 to 5 TIL subpopulations.

In some embodiments, the step of rapid second expansion is divided into multiple steps to achieve culture scale-up and scale-up by: (a) through the first container, such as G-REX- A rapid second expansion is performed by culturing TILs in small-scale cultures for a period of about 3 to 7 days in 100 MCS vessels; and then (b) transfer and distribute TILs from the small-scale cultures to at least 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers larger in size than the first, such as G-REX- Among the 500 MCS vessels, where in each second vessel, the TIL fraction from the small-scale culture transferred to such second vessel was cultured in the larger-scale culture for a period of about 4 to 7 days.

In some embodiments, the step of rapid second expansion is divided into multiple steps to achieve culture scale-up and scale-up by: (a) through the first container, such as G-REX- A rapid second expansion was performed by culturing TILs in small cultures for a period of approximately 5 days in 100 MCS vessels; and then (b) T cells from the small cultures were transferred and distributed to 2, 3 or 4 sizes In second containers that are larger than the first container, such as G-REX-500 MCS containers, wherein in each second container, the fraction of TILs from small-scale cultures that are transferred to such second containers are transferred to such second containers on a larger scale The cultures were grown for a period of about 6 days.

In some embodiments, each second container contains at least ¹⁰⁸ TILs at the time of rapid second expansion. In some embodiments, each second container comprises at least 10 ⁸ TILs, at least 10 ⁹ TILs, or at least 10 ¹⁰ TILs at the time of rapid or second expansion. In an exemplary embodiment, each second container contains at least ¹⁰¹⁰ TILs.

In some embodiments, the first small-scale TIL culture is divided into subpopulations. In some embodiments, the first small-scale TIL culture is partitioned into a plurality of about 2 to 5 subpopulations. In some embodiments, the first small-scale TIL culture is partitioned into a plurality of about 2, 3, 4, or 5 subpopulations.

In some embodiments, the plurality of subpopulations comprise a therapeutically effective amount of TILs following completion of the rapid second expansion. In some embodiments, following rapid or secondary expansion, one or more subpopulations of TILs are pooled together to produce a therapeutically effective amount of TILs. In some embodiments, each subpopulation of TILs comprises a therapeutically effective amount of TILs following rapid expansion.

In some embodiments, a rapid second amplification is performed for a period of about 3 to 7 days before dividing into a plurality of steps. In some embodiments, the germline of the rapid second amplification occurs at about day 3, day 4, day 5, day 6, day 7 after initiation of the rapid or second amplification.

In some embodiments, the splitting of the rapid second amplification occurs on about day 7, day 8, day 9, day 10, day 11 after initiation of the first amplification (i.e., pre-REP amplification) day, day 12, day 13, day 14, day 15 or day 16, day 17 or day 18. In an exemplary embodiment, the fraction of rapid or second amplification occurs about day 16 after initiation of the first amplification.

In some embodiments, the rapid second amplification is further performed for a period of about 7 to 11 days after the step. In some embodiments, the rapid second amplification is further performed for a period of about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days after the step.

In some embodiments, the cell culture medium used for the pre-step rapid second expansion comprises the same components as the cell culture medium used for the post-step rapid second expansion. In some embodiments, the cell culture medium used for the pre-step rapid second expansion comprises different components than the cell culture medium used for the post-step rapid second expansion.

In some embodiments, the cell culture medium used for rapid second expansion prior to stepping comprises IL-2, optionally OKT-3, and further optionally comprises APC. In some embodiments, the cell culture medium used for rapid second expansion before stepping comprises IL-2, OKT-3 and further optionally APC. In some embodiments, the cell culture medium used for rapid second expansion prior to fractionation comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for the rapid second expansion prior to step-up is obtained by supplementing the first expansion with fresh medium comprising IL-2, optionally OKT-3, and further optionally APCs. Proliferated cell culture medium to produce. In some embodiments, the cell culture medium used in the rapid second expansion prior to step-up is generated by supplementing the cell culture medium in the first expansion with fresh medium comprising IL-2, OKT-3, and APC. In some embodiments, the cell culture medium used for the rapid second expansion before stepping is obtained by replacing the first expansion with fresh cell culture medium comprising IL-2, optionally OKT-3, and further optionally APCs. Produced from expanding cell culture medium. In some embodiments, the cell culture medium used for the rapid second expansion prior to step-up is generated by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium for the rapid second expansion after the step comprises IL-2 and optionally OKT-3. In some embodiments, the cell culture medium for the rapid second expansion after the step comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for the post-step rapid second expansion is obtained by replacing the cell culture medium used for the pre-step rapid second expansion with fresh medium comprising IL-2 and optionally OKT-3. Increased cell culture medium to produce. In some embodiments, the cell culture medium used for the rapid second expansion after the step is obtained by replacing the cells used for the rapid second expansion before the step with fresh cell culture medium comprising IL-2 and OKT-3 culture medium to produce. 1. Feeder cells and antigen-presenting cells

In some embodiments, the rapid second amplification procedure described herein (for example comprising the following amplifications, such as in FIG. 8 (especially for example in FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. or the amplification described in step D of FIG. 8E and/or FIG. 8F and/or FIG. 8G ), and the amplification called REP) is required during REP TIL amplification and/or during rapid second amplification Excess feeder cells. In many embodiments, the feeder cell line is peripheral blood mononuclear cells (PBMC) obtained from a standard whole blood unit of a healthy blood donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation.

In general, allogeneic PBMCs were not activated by irradiation or heat treatment, and were used in the REP procedure as described in the Examples, which provide an exemplary protocol for assessing the non-replication capacity of irradiated allogeneic PBMCs.

In some embodiments, if the total number of living cells on day 7 or 14 is less than the initial living cells placed in culture on day 0 of REP and/or day 0 of the second expansion (that is, the starting day of the second expansion) number, the PBMCs were considered replication incompetent and were acceptable for use in the TIL expansion procedure described herein.

In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on day 7 and day 14 is the same as that on day 0 of REP and/or day 0 of the second expansion (i.e. PBMCs were considered to be replication incompetent and were acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng/mL OKT3 antibody and 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng/mL OKT3 antibody and 3000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on day 7 and day 14 is the same as that on day 0 of REP and/or day 0 of the second expansion (i.e. PBMCs were considered to be replication incompetent and were acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng/mL OKT3 antibody and 6000 IU/mL IL-2.

In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is about 1 to 10, about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, About 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400 or About 1 to 500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion procedure described herein requires a ratio of about 5 x ¹⁰⁸ feeder cells to about 100 x ¹⁰⁶ TILs. In some embodiments, the second expansion procedure described herein requires a ratio of about 7.5 x ¹⁰⁸ feeder cells to about 100 x ¹⁰⁶ TILs. In some embodiments, the second expansion procedure described herein requires a ratio of about 5 x ¹⁰⁸ feeder cells to about 50 x ¹⁰⁶ TILs. In some embodiments, the second expansion procedure described herein requires a ratio of about 7.5 x ¹⁰⁸ feeder cells to about 50 x ¹⁰⁶ TILs. In other embodiments, the second expansion procedure described herein requires about 5 x ¹⁰⁸ feeder cells and about 25 x ¹⁰⁶ TILs. In other embodiments, the second expansion procedure described herein requires about 7.5 x ¹⁰⁸ feeder cells and about 25 x ¹⁰⁶ TILs. In other embodiments, the rapid second expansion requires twice as many feeder cells as the rapid second expansion. In other embodiments, where about 2.5 x ¹⁰⁸ feeder cells are required for the initial first expansion described herein, about 5 x ¹⁰⁸ feeder cells are required for the rapid second expansion. In other embodiments, where about 2.5 x ¹⁰⁸ feeder cells are required for the initial first expansion described herein, about 7.5 x ¹⁰⁸ feeder cells are required for the rapid second expansion. In other embodiments, the rapid second expansion requires twice (2.0X), 2.5X, 3.0X, 3.5X, or 4.0X the number of feeder cells to initiate the first expansion.

In some embodiments, the rapid second expansion procedures described herein require an excess of feeder cells during the rapid second expansion. In many embodiments, the feeder cell line is peripheral blood mononuclear cells (PBMC) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen presenting cells (aAPCs) are used instead of PBMCs. In some embodiments, PBMCs are added to the rapid second expansion at twice the concentration of PBMCs added to initiate the first expansion.

In general, allogeneic PBMCs were inactivated by irradiation or heat treatment and used in the TIL expansion procedures described herein, including the exemplary procedures described in the Figures and Examples.

In some embodiments, artificial antigen presenting cells are used instead of or in combination with PBMCs in the rapid secondary expansion. 2. Cytokines and other additives

The rapid second expansion methods described herein typically use media with high doses of cytokines, particularly IL-2, as known in the art.

Alternatively, rapid secondary expansion of TILs is also possible using a combination of cytokines, as described in US Patent Application Publication No. US 2017/0107490 A1 using IL-2, IL-15 and IL- 21, the combination of two or more than two, the disclosure of this case is incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 as well as IL-2, IL-15 and IL-21, where the latter in many embodiments has a specific use. The use of a combination of cytokines is particularly advantageous for the generation of lymphocytes, and especially T cells as described therein.

In some embodiments, step D (particularly from, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. OKT-3 antibody or murozumab was added to the assay as described elsewhere in this article. In some embodiments, step D can also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D (particularly from, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. The OX-40 agonist was added to , as described elsewhere in this paper. Furthermore, additives may be used in the culture medium during step D (in particular from eg Fig. 8A and/or Fig. 8B and/or Fig. 8C and/or Fig. 8D and/or Fig. 8E and/or Fig. 8F and/or Fig. 8G), Such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists, such as thiazolidinedione compounds, as disclosed in U.S. Patent Application No. US 2019/0307796 A1, the disclosure of which is incorporated herein by reference.

In some embodiments, step D may also include adding protein kinase B (AKT) inhibitor (AKTi) to the culture medium. In some embodiments, the TIL population is cultured in a medium containing an AKT inhibitor to obtain a CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population. In some embodiments, the AKT inhibitor is selected from the group consisting of pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, Oridonin, gossinthin, tenoride, isoliquiritigenin, chrysanthemum, honokiol and pharmaceutically acceptable salts thereof. In some embodiments, the AKT inhibitor is pataxerti. In some embodiments, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, About 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM or about 5 μM in the culture medium of pataxerti Cultivate TIL populations. E. Step E : Collect TIL

After a rapid second expansion step, the cells can be harvested. In some embodiments, provided in, for example, FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) TILs are collected after one, two, three, four or more amplification steps. In some embodiments, provided in, for example, FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) TILs were collected after the two amplification steps. In some embodiments, provided in, for example, FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) TILs were collected after two amplification steps, an initial first amplification and a rapid second amplification.

TILs can be collected by any suitable and sterile means including, for example, centrifugation. Methods of collecting TILs are well known in the art and any such known methods may be used with the process of the invention. In some embodiments, TILs are collected using an automated system.

Cell harvesters and/or cell processing systems are commercially available from a variety of sources including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell-based harvester can be used in the methods of the invention. In some embodiments, the cell harvester and/or cell processing system is a membrane-based cell harvester. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument manufactured by any supplier that pumps a solution containing cells through a membrane or filter (such as a spin membrane or spin filter) in a sterile and/or closed system environment Or devices that allow continuous flow and cell handling to remove supernatant or cell culture medium without clumping. In some embodiments, the cell harvester and/or cell processing system can perform cell isolation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed sterile system.

In some embodiments, the rapid second amplification, for example according to FIG. 8 (especially for example FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. Step D of Fig. 8G) is performed in a closed system bioreactor. In some embodiments, TIL expansion as described herein is performed using a closed system. In some embodiments, bioreactors are employed. In some embodiments, a bioreactor is used as the container. In some embodiments, the bioreactor used is, for example, G-REX-100 or G-REX-500. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-500.

In some embodiments, step E according to FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) is Execute according to the procedure described in this article. In some embodiments, the closed system is accessed via a syringe under aseptic conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described herein is employed.

In some embodiments, TILs are collected according to the methods described herein. In some embodiments, TILs are collected between days 14 and 16 using methods as described herein. In some embodiments, TILs are collected on day 14 using methods as described herein. In some embodiments, TILs are collected on day 15 using methods as described herein. In some embodiments, TILs are collected on day 16 using methods as described herein.

In some embodiments, the invention provides for assessing or sorting the therapeutic TIL populations or TIL compositions described in the collection steps described herein for: (i) CD39/CD69 double negative and/or CD39 ^LO / CD69 ^LO , (ii) CD39/CD69 double knockout, or (iii) a combination of (i) and (ii). Methods for sorting TILs for: (i) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (ii) CD39/CD69 double gene knockout can be found in U.S. Application No. 2019/0212332 (iii) A combination of (i) and (ii), which is incorporated herein by reference. In some embodiments, cells are sorted using the method described in Zhang, X et al., "High Throughput Cell Sorting by Surface Free Energy Activation", "Analytical Chemistry" (2014), 86: 9350-9355. Example 15 and Figure 41 provide examples of sorting schemes using such methods. F. Step F : Final formulation and transfer to infusion container

8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. After completion of Steps A through E as outlined in detail herein, the cells are transferred to a container for administration to the patient, such as an infusion bag or sterile vial. In some embodiments, once therapeutically sufficient numbers of TILs are obtained using the expansion methods described above, they are transferred to containers for administration to a patient.

In some embodiments, TILs expanded using the methods of the disclosure are administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TIL in a sterile buffer. TILs expanded as disclosed herein can be administered by any suitable route known in the art. In some embodiments, TIL is administered as a single intra-arterial or intravenous infusion, which preferably lasts for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

In some embodiments, the present invention provides methods as described in any preceding paragraph, as applicable, such that (a) prior to initiating the first amplification (i), in the presence of IL-2 and optionally a first antibiotic culturing the bulk TIL or first TIL population in the tumor fragment or sample in a combined cell culture medium to produce TIL released from the tumor fragment or sample, (ii) isolating from the tumor fragment or sample at least a plurality of TILs released from the tumor fragment or sample TILs released from the tumor fragment or sample to produce a mixture of tumor fragment or sample, residual TIL in the tumor fragment or sample, and any TIL released from the tumor fragment or sample and remaining with it after isolation, and (iii) optionally separating the tumor fragment or sample or a mixture of TIL remaining in the sample, tumor fragment or sample, and any TIL released from the tumor fragment or sample and remaining with it after isolation to produce a digest of such mixture; and (b) using the mixture or mixture The digest was carried out to initiate the first amplification. In some embodiments, at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 95%, 99% or higher percentage of TILs released from tumor fragments or samples are isolated from the tumor fragments or samples to produce a mixture.

In some embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified such that the culturing step prior to initiating the first amplification is performed for a period of about 1 day to about 3 days.

In some embodiments, the invention provides a method as described in any preceding paragraph as applicable above, modified such that the culturing step performed prior to initiating the first amplification is performed for about 1, 2, 3, 4, 5 , 6 or 7 days.

In some embodiments, the present invention provides a method as described in any of the preceding paragraphs where applicable such that (a) a CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO preselection step prior to initiating the first amplification Prior to (i) culturing the bulk TIL or first TIL population in the tumor fragment or sample in cell culture medium containing IL-2 to produce TIL released from the tumor fragment or sample, (ii) isolating from the tumor fragment or sample at least a plurality of TILs released from the tumor fragment or sample to produce a mixture of the tumor fragment or sample, residual TIL in the tumor fragment or sample, and any TIL released from the tumor fragment or sample and remaining with it after isolation, and (iii) digesting a mixture of the tumor fragment or sample, TIL remaining in the tumor fragment or sample, and any TIL released from the tumor fragment or sample and remaining with it after isolation to produce a digest of such mixture; and ( b) A CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO pre-selection step was performed using a digest of the mixture to generate a CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population. In some embodiments, at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 95%, 99% or higher percentage of TILs released from tumor fragments or samples are isolated from the tumor fragments or samples to produce a mixture.

In some embodiments, the invention provides a method as described in any preceding paragraph as applicable above such that the culturing step prior to the CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO preselection step is performed for about 1 day to about 3 days time of day.

In some embodiments, the invention provides a method as described in any preceding paragraph as applicable, such that the culturing step prior to the CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO preselection step is performed for about 1, 2, 3 , 4, 5, 6 or 7 day period. V. Other Gen 2 , Gen 3 and other TIL manufacturing process examples A. PBMC feeder cell ratio

In some embodiments, for the amplification methods described herein (see, for example, FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. The medium in 8F and/or FIG. 8G)) includes an anti-CD3 antibody, eg OKT-3. Combination of anti-CD3 antibody and IL-2 induces T cell activation and cell division in TIL populations. This effect is seen for full length antibodies as well as Fab and F(ab')2 fragments, the former generally being preferred; see eg Tsoukas et al., J. Immunology 1985, 135 , 1719, which is hereby incorporated by reference in its entirety.

In some embodiments, the number of PBMC feeder cell layers is calculated as follows: T cell volume (10 µm in diameter): V = (4/3) πr ³ =523.6 µm ³ G-REX with a height of 40 µm (4 cells) -100(M) column: V = (4/3) πr ³ = 4×10 ¹² µm ³ The number of cells required to fill column B: 4×10 ¹² µm ³ /523.6 µm ³ = 7.6×10 ⁸ µm ³ ×0.64 = 4.86×10 ⁸ Number of cells that can be optimally activated in 4D space: 4.86×10 ⁸ /24 = 20.25×10 ⁶ Number of feeder cells and TIL extrapolated to G-Rex-500: TIL: 100×10 ⁶ and feeder cells: 2.5×10 ⁹

In this calculation, the approximate number of monocytes required to provide icosahedral geometry for TIL activation in a cylinder with a 100 ^cm base was used. The calculated experimental result of T cell threshold activation is about 5×10 ⁸ , which is closely related to the NCI experimental data, such as Jin et al., "Journal of Immunotherapy", 2012, 35, 283-292. In (C), the multiplier (0.64) is the random packing density of the equivalent sphere, calculated by Jaeger and Nagel, Science , 1992, 255 , 1523-3. In (D), the divisor 24 is the number of equivalent spheres or "Newton numbers" that can touch similar objects in 4-dimensional space, such as Musin, "Russian Mathematical Review ( Russ. Math. Surv. ), 2003, 58 , 794-795 as described.

In some embodiments, the number of antigen-presenting feeder cells exogenously supplied during the initial first expansion is about half the number of antigen-presenting feeder cells exogenously supplied during the rapid second expansion. In certain embodiments, the method comprises performing the initial first expansion in a cell culture medium comprising about 50% fewer antigen-presenting cells than the cell culture medium of the rapid second expansion.

In other embodiments, the number of antigen-presenting feeder cells (APCs) exogenously supplied during the rapid second expansion is greater than the number of APCs exogenously supplied during the initial first expansion.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 20 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to just at or about 10 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly at or about 1.1:1 to just at or about 9 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 8 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly at or about 1.1:1 to just at or about 7 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 6 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 5 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 4 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 3 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.9 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly at or about 1.1:1 to exactly at or about 2.8 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.7 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly at or about 1.1:1 to exactly at or about 2.6 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly at or about 1.1:1 to exactly at or about 2.5 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.4 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly at or about 1.1:1 to exactly at or about 2.3 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly at or about 1.1:1 to just at or about 2.2 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.1 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to just at or about 10 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to exactly or about 5 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to exactly or about 4 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to exactly or about 3 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.9 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.8 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.7 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.6 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.5 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.4 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.3 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly at or about 2:1 to just at or about 2.2 :1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.1 :1 range.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is exactly or about 2:1.

In other embodiments, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is exactly or about 1.1:1, 1.2:1, 1.3: 1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8: 1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

In other embodiments, the number of exogenously supplied APCs during the initiation of the first amplification is exactly or about 1×10 ⁸ , 1.1×10 ⁸ , 1.2×10 ⁸ , 1.3×10 ⁸ , 1.4×10 ⁸ , 1.5 ×10 ⁸ , 1.6×10 ⁸ , 1.7×10 ⁸ , 1.8×10 ^{8 , 1.9×10 8} , 2×10 ⁸ , 2.1×10 ⁸ , 2.2×10 ⁸ , 2.3× ^{10 8 ,} 2.4×10 ⁸ , 2.5 ×10 ⁸ , 2.6×10 ⁸ , 2.7×10 ⁸ , 2.8×10 ⁸ , 2.9×10 ⁸ , 3×10 ⁸ , 3.1×10 ⁸ , 3.2×10 ⁸ , 3.3×10 ⁸ , 3.4×10 ⁸ or 3.5 ×10 ⁸ APCs, and the number of exogenously supplied APCs during the rapid second amplification was just or about 3.5×10 ⁸ , 3.6×10 ⁸ , 3.7×10 ⁸ , 3.8×10 ⁸ , 3.9×10 ⁸ , 4 ×10 ⁸ , 4.1×10 ⁸ , 4.2×10 ⁸ , 4.3×10 ⁸ , 4.4×10 ⁸ , 4.5×10 ⁸ , 4.6×10 ⁸ , 4.7×10 ⁸ , 4.8×10 ⁸ , 4.9×10 ⁸ , 5 ×10 ⁸ , 5.1×10 ⁸ , 5.2×10 ⁸ , 5.3×10 ^{8 , 5.4×10 8} , 5.5×10 ⁸ , 5.6×10 ⁸ , 5.7×10 ⁸ , 5.8×10 ⁸ , 5.9× ^{10 8 ,} 6 ×10 ⁸ , 6.1×10 ⁸ , 6.2×10 ⁸ , 6.3×10 ⁸ , 6.4×10 ⁸ , 6.5×10 ⁸ , 6.6×10 ⁸ , 6.7×10 ⁸ , 6.8×10 ⁸ , 6.9×10 ⁸ , 7 ×10 ⁸ , 7.1×10 ⁸ , 7.2×10 ⁸ , 7.3×10 ^{8 , 7.4×10 8} , 7.5×10 ⁸ , 7.6×10 ⁸ , 7.7×10 ⁸ , 7.8×10 ⁸ , 7.9×10 ⁸ , ⁸ ×10 ⁸ , 8.1×10 ⁸ , 8.2×10 ⁸ , 8.3×10 8 , 8.4×10 ⁸ , 8.5×10 ⁸ , 8.6×10 ⁸ , 8.7×10 ⁸ , 8.8× ^{10 8 ,} 8.9×10 ⁸ , 9 ×10 ⁸ , 9.1×10 ⁸ , 9.2×10 ⁸ , 9.3×10 ⁸ , 9.4×10 ⁸ , 9.5×10 ⁸ , 9.6×10 ⁸ , 9.7×10 ⁸ , 9.8×10 ⁸ , 9.9×10 ⁸ or 1 ×10 ⁹ APCs.

In other embodiments, the number of APCs exogenously supplied during the initial first amplification is selected from the range of exactly or about 1.5 x ¹⁰⁸ APCs to exactly or about 3 x ¹⁰⁸ APCs, and during the rapid second The number of APCs supplied exogenously during expansion is selected from the range of just or about 4 x ¹⁰⁸ APCs to just or about 7.5 x ¹⁰⁸ APCs.

In other embodiments, the number of APCs exogenously supplied during the initial first amplification is selected from the range of exactly or about 2× ¹⁰ APCs to exactly or about 2.5× ¹⁰ APCs, and during the rapid second The number of APCs supplied exogenously during expansion is selected from the range of just or about 4.5 x ¹⁰⁸ APCs to just or about 5.5 x ¹⁰⁸ APCs.

In other embodiments, the number of APCs exogenously supplied during the initial first amplification is exactly or about 2.5 x ¹⁰⁸ APCs, and the number of APCs exogenously supplied during the rapid second amplification is exactly or about 5 × ¹⁰⁸ APCs.

In some embodiments, the number of APCs (including, e.g., PBMCs) added on Day 0 of initiation of the first expansion is approximately the number of PBMCs added on Day 7 of initiation of the first expansion (e.g., Day 7 of the method). half. In certain embodiments, the method comprises adding antigen presenting cells to a first population of TILs on day 0 of initiating the first expansion and adding antigen presenting cells to a second population of TILs on day 7, wherein the addition on day 0 The number of antigen-presenting cells is approximately 50% of the number of antigen-presenting cells added on day 7 of initiation of the first expansion (eg, day 7 of the method).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion is greater than the number of exogenously supplied PBMCs on day 0 of the initiation of the first expansion.

In other embodiments, the exogenously supplied APCs at the initiation of the first expansion are selected from the range of exactly or about 1.0×10 ⁶ APCs/cm ² to exactly or about 4.5×10 ⁶ APCs/cm ² Density seeding in culture flasks.

In other embodiments, the exogenously supplied APCs at the initiation of the first expansion are selected from within the range of exactly or about 1.5 x ¹⁰⁶ APCs/ ^cm2 to exactly or about 3.5 x ¹⁰⁶ APCs/ ^cm2 Density seeding in culture flasks.

In other embodiments, the exogenously supplied APCs at the initiation of the first expansion are selected from within the range of exactly or about 2×10 ⁶ APCs/cm ² to exactly or about 3×10 ⁶ APCs/cm ² Density seeding in culture flasks.

In other embodiments, exogenously supplied APCs are seeded in culture flasks at a density of just or about 2 x ¹⁰⁶ APCs/ ^cm2 at the initiation of the first expansion.

In other embodiments, the APCs are exogenously supplied at the initiation of the first amplification at just or about 1.0×10 ⁶ , 1.1×10 ⁶ , 1.2×10 ⁶ , 1.3×10 ⁶ , 1.4×10 ⁶ , 1.5×10 6 10 ⁶ , 1.6×10 ⁶ , 1.7×10 ⁶ , 1.8×10 6 , 1.9×10 ⁶ , 2×10 ⁶ , 2.1×10 ⁶ , 2.2×10 ⁶ , 2.3× ^{10 6 ,} 2.4×10 ⁶ , 2.5× 10 ⁶ , 2.6×10 ⁶ , 2.7×10 ⁶ , 2.8×10 ⁶ , 2.9×10 ⁶ , 3×10 ⁶ , 3.1×10 ⁶ , 3.2×10 ⁶ , 3.3×10 ⁶ , 3.4×10 ⁶ , 3.5× 10 ⁶ , 3.6×10 ⁶ , 3.7×10 ⁶ , 3.8×10 ⁶ , 3.9×10 ⁶ , 4×10 ⁶ , 4.1×10 ⁶ , 4.2×10 ⁶ , 4.3×10 ⁶ , 4.4×10 ⁶ or 4.5× The density of 10 ⁶ APC/cm ² was inoculated in the culture flask.

In other embodiments, the exogenously supplied APCs in the rapid second expansion are at a density selected from the range of just or about 2.5 x ¹⁰⁶ APCs/ ^cm2 to just or about 7.5 x ¹⁰⁶ APCs/ ^cm2 Inoculated into culture flasks.

In other embodiments, the exogenously supplied APCs in the rapid second expansion are at a density selected from the range of just or about 3.5 x ¹⁰⁶ APCs/ ^cm2 to just or about 6.0 x ¹⁰⁶ APCs/ ^cm2 Inoculated into culture flasks.

In other embodiments, the exogenously supplied APCs in the rapid second expansion are at a density selected from the range of just or about 4.0 x ¹⁰⁶ APCs/ ^cm2 to just or about 5.5 x ¹⁰⁶ APCs/ ^cm2 Inoculated into culture flasks.

In other embodiments, exogenously supplied APCs in the rapid second expansion are seeded in culture flasks at a density selected from the range of just or about 4.0 x ¹⁰⁶ APCs/ ^cm2 .

In other embodiments, the exogenously supplied APC line is expanded at just or about 2.5×10 ⁶ APC/cm ² , 2.6×10 ⁶ APC/cm ² , 2.7×10 ⁶ APC/cm 2 during rapid second expansion. ² , 2.8×10 ⁶ , 2.9×10 ⁶ , 3×10 ⁶ , 3.1×10 6 , 3.2×10 ⁶ , 3.3×10 ⁶ , 3.4×10 ⁶ , 3.5×10 ⁶ , 3.6× ^{10 6 ,} 3.7×10 6 ⁶ , 3.8×10 ⁶ , 3.9×10 ⁶ , 4×10 6 , 4.1×10 ⁶ , 4.2×10 ⁶ , 4.3×10 ⁶ , 4.4×10 ⁶ , 4.5×10 ⁶ , 4.6× ^{10 6 ,} 4.7×10 6 ⁶ , 4.8×10 ⁶ , 4.9×10 ⁶ , 5×10 6 , 5.1×10 ⁶ , 5.2×10 ⁶ , 5.3×10 ⁶ , 5.4×10 ⁶ , 5.5×10 ⁶ , 5.6× ^{10 6 ,} 5.7×10 ⁶ , 5.8×10 ⁶ , 5.9×10 ⁶ , 6×10 6 , 6.1×10 ⁶ , 6.2×10 ⁶ , 6.3×10 ⁶ , 6.4×10 ⁶ , 6.5×10 ⁶ , 6.6× ^{10 6 ,} 6.7×10 6 ⁶ , 6.8×10 ⁶ , 6.9×10 ⁶ , 7×10 ⁶ , 7.1×10 ^{6 , 7.2×10 6 ,} 7.3×10 ⁶ , 7.4×10 ⁶ or 7.5×10 ⁶ APC/cm ² were inoculated on in a culture bottle.

In other embodiments, the exogenously supplied APCs in the initial first amplification are at or about 1.0×10 ⁶ , 1.1×10 ⁶ , 1.2×10 ⁶ , 1.3×10 ⁶ , 1.4×10 ⁶ , 1.5×10 6 10 ⁶ , 1.6×10 ⁶ , 1.7×10 ⁶ , 1.8×10 6 , 1.9×10 ⁶ , 2×10 ⁶ , 2.1×10 ⁶ , 2.2×10 ⁶ , 2.3× ^{10 6 ,} 2.4×10 ⁶ , 2.5× 10 ⁶ , 2.6×10 ⁶ , 2.7×10 ⁶ , 2.8×10 ⁶ , 2.9×10 ⁶ , 3×10 ⁶ , 3.1×10 ⁶ , 3.2×10 ⁶ , 3.3×10 ⁶ , 3.4×10 ⁶ , 3.5× 10 ⁶ , 3.6×10 ⁶ , 3.7×10 ⁶ , 3.8×10 ⁶ , 3.9×10 ⁶ , 4×10 ⁶ , 4.1×10 ⁶ , 4.2×10 ⁶ , 4.3×10 ⁶ , 4.4×10 ⁶ or 4.5× Flasks were seeded at a density of 10 ⁶ APCs/cm ² and exogenously supplied APCs in the rapid second expansion at just or about 2.5×10 ⁶ APCs/cm ² , 2.6×10 ⁶ APCs/cm 2 ² , 2.7×10 ⁶ APC/cm ² , 2.8×10 ⁶ , 2.9×10 ⁶ , 3×10 ⁶ , 3.1×10 ⁶ , 3.2×10 ⁶ , 3.3×10 ⁶ , 3.4×10 ⁶ , 3.5×10 ⁶ , 3.6×10 ⁶ , 3.7×10 ⁶ , 3.8×10 ⁶ , 3.9×10 ⁶ , 4×10 ⁶ , 4.1×10 ⁶ , 4.2×10 ⁶ , 4.3×10 ⁶ , 4.4×10 ⁶ , 4.5×10 6 ⁶ , 4.6×10 ⁶ , 4.7×10 ^{6 , 4.8×10 6 ,} 4.9×10 ⁶ , 5×10 ⁶ , 5.1×10 ⁶ , 5.2×10 ⁶ , 5.3×10 ⁶ , 5.4×10 ⁶ , 5.5×10 6 ⁶ , 5.6×10 ⁶ , 5.7×10 ^{6 , 5.8×10 6} , 5.9×10 ⁶ , 6×10 ⁶ , 6.1×10 ⁶ , 6.2×10 ⁶ , 6.3×10 ⁶ , 6.4× ^{10 6 ,} 6.5×10 6 ⁶ , 6.6×10 ⁶ , 6.7×10 ⁶ , 6.8×10 6 , 6.9×10 ⁶ , 7×10 ⁶ , 7.1×10 ⁶ , 7.2×10 ⁶ , 7.3×10 ⁶ , 7.4× ^{10 6} or 7.5×10 6 The density of ⁶ APC/cm ² was inoculated in the culture flask.

In other embodiments, the exogenously supplied APCs at the initiation of the first expansion are in the range selected from exactly or about 1.0 x ¹⁰⁶ APCs/ ^cm2 to exactly or about 4.5 x ¹⁰⁶ APCs/ ^cm2 The density was seeded in culture flasks, and in the rapid second expansion the exogenously supplied APC line was selected from the range of just or about 2.5 x ¹⁰⁶ APCs/ ^cm2 to just or about 7.5 x ¹⁰⁶ APCs/ ^cm2 Inoculate the culture flask at a density of .

In other embodiments, the exogenously supplied APCs at the initiation of the first expansion are selected from the range of exactly or about 1.5 x ¹⁰⁶ APCs/ ^cm2 to exactly or about 3.5 x ¹⁰⁶ APCs/ ^cm2 The density was seeded in culture flasks, and in the rapid second expansion the exogenously supplied APC line was selected from the range of just or about 3.5 x ¹⁰⁶ APCs/ ^cm2 to just or about 6 x ¹⁰⁶ APCs/ ^cm2 Inoculate the culture flask at a density of .

In other embodiments, the exogenously supplied APCs at the initiation of the first expansion are selected from within the range of exactly or about 2×10 ⁶ APCs/cm ² to exactly or about 3×10 ⁶ APCs/cm ² The density was seeded in culture flasks, and in the rapid second expansion the exogenously supplied APC line was selected from the range of just or about 4 x ¹⁰⁶ APCs/ ^cm2 to just or about 5.5 x ¹⁰⁶ APCs/ ^cm2 Inoculate the culture flask at a density of .

In other embodiments, exogenously supplied APCs are seeded in flasks at a density of just or about 2 x ¹⁰⁶ APCs/ ^cm2 at the initial first expansion, and exogenously supplied APCs at the rapid second expansion The APCs were seeded in culture flasks at a density of just or about 4×10 ⁶ APCs/cm ² .

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied PBMCs on day 0 of the initiation of the first expansion is selected from exactly or about 1.1 :1 to exactly or about 20:1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied PBMCs on day 0 of the initiation of the first expansion is selected from exactly or about 1.1 :1 to exactly or about 10:1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied PBMCs on day 0 of the initiation of the first expansion is selected from exactly or about 1.1 :1 to exactly or about 9:1 range.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 1.1:1 to just or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from exactly or about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected Range from exactly or about 1.1:1 to exactly or about 2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 2:1 to just or about 10:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 2:1 to just or about 5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 2:1 to just or about 4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 2:1 to just or about 3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from exactly or about 2:1 to exactly or about 2.9:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 2:1 to just or about 2.8:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 2:1 to just or about 2.7:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 2:1 to just or about 2.6:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 2:1 to just or about 2.5:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 2:1 to just or about 2.4:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from just or about 2:1 to just or about 2.3:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from exactly or about 2:1 to exactly or about 2.2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is selected A range from exactly or about 2:1 to exactly or about 2.1:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is just right Or about 2:1.

In other embodiments, the ratio of the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion to the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion is just right or approximately 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5: 1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1 or 5:1.

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of initiation of the first expansion is exactly or about 1×10 ⁸ , 1.1×10 ⁸ , 1.2×10 ⁸ , 1.3×10 ⁸ , 1.4×10 ⁸ , 1.5×10 ⁸ , 1.6×10 8 , 1.7×10 ⁸ , 1.8×10 ⁸ , 1.9×10 ⁸ , 2×10 ⁸ , 2.1×10 ⁸ , 2.2× ^{10 8 ,} 2.3×10 ⁸ , 2.4 ^× 10 ⁸ , 2.5×10 ⁸ , 2.6×10 ^{8 , 2.7×10 8} , 2.8×10 ⁸ , 2.9×10 ⁸ , 3×10 ⁸ , 3.1×10 ⁸ , 3.2×10 ⁸ , 3.3×10 ⁸ , 3.4×10 ⁸ or 3.5×10 ⁸ APCs (including, for example, PBMCs), and the number of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion was just or about 3.5×10 ⁸ , 3.6×10 ⁸ , 3.7×10 ⁸ , 3.8×10 ⁸ , 3.9×10 ⁸ , 4×10 ⁸ , 4.1×10 8 , 4.2×10 ⁸ , 4.3×10 ⁸ , 4.4×10 ⁸ , 4.5× ^{10 8 ,} 4.6×10 ⁸ , 4.7×10 ⁸ , 4.8×10 ⁸ , 4.9×10 ⁸ , 5×10 ⁸ , 5.1×10 8 , 5.2×10 ⁸ , 5.3×10 ⁸ , 5.4×10 ⁸ , 5.5× ^{10 8 ,} 5.6×10 ⁸ , 5.7×10 ⁸ , 5.8×10 ⁸ , 5.9×10 ⁸ , 6×10 8 , 6.1×10 ⁸ , 6.2×10 ⁸ , 6.3×10 ⁸ , 6.4×10 ⁸ , 6.5× ^{10 8 ,} 6.6×10 ⁸ , 6.7×10 ⁸ , 6.8×10 ⁸ , 6.9×10 ⁸ , 7×10 8 , 7.1×10 ⁸ , 7.2×10 ⁸ , 7.3×10 ⁸ , 7.4×10 ⁸ , 7.5× ^{10 8 ,} 7.6×10 ⁸ , 7.7×10 ⁸ , 7.8×10 ⁸ , 7.9×10 ⁸ , 8×10 8 , 8.1×10 ⁸ , 8.2×10 ⁸ , 8.3×10 ⁸ , 8.4×10 ⁸ , 8.5× ^{10 8 ,} 8.6×10 ⁸ , 8.7×10 ⁸ , 8.8×10 ⁸ , 8.9×10 ⁸ , 9×10 8 , 9.1×10 ⁸ , 9.2×10 ⁸ , 9.3×10 ⁸ , 9.4×10 ⁸ , 9.5× ^{10 8 ,} 9.6×10 ⁸ , 9.7×10 ⁸ , 9.8×10 ⁸ , 9.9×10 ⁸ or 1×10 ⁹ APCs (including for example P BMC).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of initiation of the first expansion is selected from exactly or about 1 x ¹⁰ APCs (including, for example, PBMCs) to just or about 3.5 x 10 The range of ⁸ APCs (including, for example, PBMCs) and the number of exogenously supplied APCs (including, for example, PBMCs) at day 7 of the rapid second expansion is selected from exactly or about 3.5 x ¹⁰⁸ APCs (including, for example, PBMCs) to just Or a range of about 1 x ¹⁰⁹ APCs (including eg PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) at the initiation of the first expansion on day 0 is selected from exactly or about 1.5 x ¹⁰⁸ APCs to exactly or about 3 x ¹⁰⁸ APCs (including e.g. PBMC) and the number of exogenously supplied APCs (including e.g. PBMCs) at day 7 of the rapid second expansion is selected from just or about 4 x ¹⁰ APCs (including e.g. PBMCs) to just or about 7.5 x 10 Range of ⁸ APCs including eg PBMCs.

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) on day 0 of initiation of the first expansion is selected from exactly or about 2×10 ^APCs (including, for example, PBMCs) to exactly or about 2.5×10 The range of ⁸ APCs (including, for example, PBMCs) and the number of exogenously supplied APCs (including, for example, PBMCs) at day 7 of the rapid second expansion is selected from exactly or about 4.5 x ¹⁰⁸ APCs (including, for example, PBMCs) to just Or a range of about 5.5 x ¹⁰⁸ APCs (including eg PBMCs).

In other embodiments, the number of exogenously supplied APCs (including, for example, PBMCs) at day 0 of initiation of the first expansion is exactly or about 2.5 x ¹⁰⁸ APCs (including, for example, PBMCs) and at day 0 of the rapid second expansion The number of exogenously supplied APCs (including eg PBMCs) at 7 days was exactly or about 5 x ¹⁰⁸ APCs (including eg PBMCs).

In some embodiments, the layer of APCs (including, for example, PBMCs) added on day 0 of the initial first expansion is about half the number of layers of APCs (including, for example, PBMCs) added on day 7 of the rapid second expansion. In certain embodiments, the method comprises adding the antigen-presenting cell layer to the first TIL population on day 0 of initiating the first expansion and adding the antigen-presenting cell layer to the second TIL population on day 7, wherein on day 0 The number of antigen-presenting cell layers added was about 50% of the number of antigen-presenting cell layers added on day 7.

In other embodiments, the layer of exogenously supplied APCs (including, for example, PBMCs) on day 7 of the rapid second expansion is greater than the number of layers of exogenously supplied APCs (including, for example, PBMCs) on day 0 of the initiation of the first expansion.

In other embodiments, day 0 of initiation of the first expansion occurs in the presence of lamellar APCs (including, for example, PBMCs) with an average thickness of just or about 2 cell layers, and day 7 of rapid second expansion occurs at Occurs in the presence of lamellar APCs (including eg PBMCs) with an average thickness of just or about 4 cell layers.

In other embodiments, day 0 of initiation of the first expansion occurs in the presence of lamellar APCs (including, for example, PBMCs) with an average thickness of just or about one cell layer, and day 7 of rapid second expansion occurs on average Occurs in the presence of lamellar APCs (including, for example, PBMCs) just or about 3 cell layers thick.

In other embodiments, day 0 of initiating the first expansion occurs in the presence of lamellar APCs (including, for example, PBMCs) having an average thickness of just or about 1.5 cell layers to just or about 2.5 cell layers, and the rapid day 0 Day 7 of the second expansion occurs in the presence of lamellar APCs (including eg PBMCs) with an average thickness of just or about 3 cell layers.

In other embodiments, day 0 of initiation of the first expansion occurs in the presence of lamellar APCs (including, for example, PBMCs) with an average thickness of just or about one cell layer, and day 7 of rapid second expansion occurs on average Occurs in the presence of lamellar APCs (including, for example, PBMCs) just or about 2 cell layers thick.

In other embodiments, day 0 of initiating the first expansion is at or at an average thickness of about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, Occurs in the presence of 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers of lamellar APCs (including, for example, PBMCs) and the day 7 of rapid second expansion occurs at an average thickness of at or about 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6 , 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8 cells Layered lamellar APCs (including, for example, PBMCs) occur in the presence.

In other embodiments, day 0 of initiation of the first expansion occurs in the presence of lamellar APCs (including, for example, PBMCs) with an average thickness of just or about 1 cell layer to just or about 2 cell layers, and the rapid day 0 Day 7 of the second expansion occurs in the presence of lamellar APCs (including, for example, PBMCs) with an average thickness of just or about 3 cell layers to just or about 10 cell layers.

In other embodiments, day 0 of initiation of the first expansion occurs in the presence of lamellar APCs (including, for example, PBMCs) with an average thickness of just or about 2 cell layers to just or about 3 cell layers, and the rapid day 0 Day 7 of the second expansion occurs in the presence of lamellar APCs (including, for example, PBMCs) with an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, day 0 of initiation of the first expansion occurs in the presence of lamellar APCs (including, for example, PBMCs) with an average thickness of just or about 2 cell layers, and day 7 of rapid second expansion occurs at Occurs in the presence of lamellar APCs (including, for example, PBMCs) having an average thickness of just or about 4 cell layers to just or about 8 cell layers.

In other embodiments, day 0 of initiation of the first expansion occurs in the presence of lamellar APCs (including, for example, PBMCs) with an average thickness of just or about 1, 2, or 3 cell layers, and rapid second expansion. Day 7 occurs in the presence of lamellar APCs (including, for example, PBMCs) having an average thickness of just or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.1 to just or about 1:10.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.1 to just or about 1:8.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.1 to just or about 1:7.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.1 to just or about 1:6.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.1 to just or about 1:5.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.1 to just or about 1:4.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.1 to just or about 1:3.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.1 to just or about 1:2.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.2 to just or about 1:8.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.3 to just or about 1:7.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.4 to just or about 1:6.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.5 to just or about 1:5.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.6 to just or about 1:4.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.7 to just or about 1:3.5.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.8 to just or about 1:3.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is selected from the range of just or about 1:1.9 to just or about 1:2.5.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising eg PBMC) is exactly or about 1:2.

In other embodiments, the day 0 of initiating the first expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a first average thickness equal to the number of layers of the first APC (including, for example, PBMCs), and the rapid second Day 7 of the second expansion occurs in the presence of layered APCs (including, for example, PBMCs) having a second average thickness equal to the second layer of APCs (including, for example, PBMCs), wherein the first layer of APCs (including, for example, PBMCs) is equal to The ratio of the number of layers of the second APC (comprising e.g. PBMC) is selected from exactly or about 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1: 3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1: 5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7, 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:7.9 8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, or 1:10.

In some embodiments, the number of APCs in the initial first expansion is selected from the range of about 1.0×10 ⁶ APCs/cm ² to about 4.5×10 ⁶ APCs/cm ² , and the number of APCs in the rapid second expansion The number of APCs is selected from the range of about 2.5×10 ⁶ APCs/cm ² to about 7.5×10 ⁶ APCs/cm ² .

In some embodiments, the number of APCs in the initial first expansion is selected from the range of about 1.5×10 ⁶ APCs/cm ² to about 3.5×10 ⁶ APCs/cm ² , and the number of APCs in the rapid second expansion The number of APCs is selected from the range of about 3.5×10 ⁶ APC/cm ² to about 6.0×10 ⁶ APC/cm ² .

In some embodiments, the number of APCs in the initial first expansion is selected from the range of about 2.0×10 ⁶ APCs/cm ² to about 3.0×10 ⁶ APCs/cm ² , and the number of APCs in the rapid second expansion is The number of APCs is selected from the range of about 4.0×10 ⁶ APC/cm ² to about 5.5×10 ⁶ APC/cm ² . B. Components of cell culture medium selected according to the situation 1. Anti-CD3 antibody

In some embodiments, the medium used in the expansion methods described herein (see, eg, Figures 1 and 8 (in particular, eg, Figure 8B)) includes an anti-CD3 antibody. Combination of anti-CD3 antibody and IL-2 induces T cell activation and cell division in TIL populations. This effect is seen for full length antibodies as well as Fab and F(ab')2 fragments, the former generally being preferred; see eg Tsoukas et al., J. Immunology 1985, 135 , 1719, which is hereby incorporated by reference in its entirety.

Those skilled in the art will appreciate that a number of suitable anti-human CD3 antibodies may be used in the present invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including but not limited to murine, human, spiritual Longine, rat and canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody murozumab (available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotechnology, Auburn, CA) is used. See Table 1.

Those skilled in the art will appreciate that a number of suitable anti-human CD3 antibodies may be used in the present invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including but not limited to murine, human, spiritual Longine, rat and canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody murozumab (available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotechnology, Auburn, CA) is used. 2. 4-1BB(CD137) agonists

In some embodiments, the cell culture medium that initiates the first expansion and/or the rapid second expansion comprises a TNFRSF agonist. In some embodiments, the TNFRSF agonist is a 4-1BB(CD137) agonist. The 4-1BB agonist can be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule can be a monoclonal antibody or a fusion protein capable of binding to human or mammalian 4-1BB. A 4-1BB agonist or 4-1BB binding molecule may comprise any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of an immunoglobulin molecule ) or a subclass of immunoglobulin heavy chain. A 4-1BB agonist or 4-1BB binding molecule can have a heavy chain and a light chain. As used herein, the term binding molecule also includes antibodies (including full-length antibodies); monoclonal antibodies (including full-length monoclonal antibodies); polyclonal antibodies; multispecific antibodies (such as bispecific antibodies); Chimeric antibodies; and antibody fragments, such as Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the foregoing, and engineered forms of antibodies that bind 4-1BB , such as scFv molecules. In some embodiments, the 4-1BB agonist is an antigen binding protein of a fully human antibody. In some embodiments, the 4-1BB agonist is an antigen binding protein of a humanized antibody. In some embodiments, 4-1BB agonists used in the methods and compositions disclosed herein include anti-4-1BB antibodies, human anti-4-1BB antibodies, mouse anti-4-1BB antibodies, mammalian anti-4-1BB antibodies, 4-1BB antibody, monoclonal anti-4-1BB antibody, polyclonal anti-4-1BB antibody, chimeric anti-4-1BB antibody, anti-4-1BB adnectin, anti-4-1BB domain antibody, single chain Anti-4-1BB fragments, heavy chain anti-4-1BB fragments, light chain anti-4-1BB fragments, anti-4-1BB fusion proteins, and fragments, derivatives, conjugates, variants or biosimilars thereof. Potent anti-4-1BB antibodies are known to induce strong immune responses. Lee et al., PLOS One 2013 , 8, e69677. In some embodiments, the 4-1BB agonist is an agonistic anti-4-1BB humanized or fully human monoclonal antibody (ie, an antibody derived from a single cell line). In some embodiments, the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.), Utumumab or Urelumab or a fragment, derivative, conjugate, variant or biosimilar thereof things. In some embodiments, the 4-1BB agonist is utumumab or uselumab or a fragment, derivative, conjugate, variant or biosimilar thereof.

In some embodiments, the 4-1BB agonist or 4-1BB binding molecule can also be a fusion protein. In some embodiments, a polymeric 4-1BB agonist, such as a trimeric or hexameric 4-1BB agonist (having three or six ligand-binding domains) can induce clustering of elite receptors (4-1BBL) and formation of internal cell-messaging complexes. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linking two or more of these fusion proteins are described, for example, in Gieffers et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

Potent 4-1BB antibodies and fusion proteins are known to induce strong immune responses. In some embodiments, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to the 4-1BB antigen in a manner sufficient to reduce toxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates antibody-dependent cellular cytotoxicity (ADCC), such as NK cell toxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abolishes antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abolishes complement dependent cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abolishes Fc region functionality.

In some embodiments, the 4-1BB agonist is characterized as binding to human 4-1BB (SEQ ID NO: 40) with high affinity and agonist activity. In some embodiments, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO: 40). In some embodiments, the 4-1BB agonist is a binding molecule that binds to murine 4-1BB (SEQ ID NO: 41). The amino acid sequences of the 4-1BB antigens to which the 4-1BB agonists or binding molecules bind are summarized in Table 5.

In some embodiments, the compositions, procedures and methods described include a 4-1BB agonist that binds human or murine 4-1BB with a KD of about 100 pM or less, with a KD of about 100 pM or less. Binds human or murine 4-1BB with a KD of 90 pM or less, binds human or murine 4-1BB with a KD of about 80 pM or less, binds human or murine 4-1BB with a KD of about 70 pM or less 1BB, binds human or murine 4-1BB with a KD of about 60 pM or less, binds human or murine 4-1BB with a KD of about 50 pM or less, binds human or murine 4-1BB with a KD of about 40 pM or less Murine 4-1BB, or binds human or murine 4-1BB with a KD of about 30 pM or less.

In some embodiments, the described compositions, processes and methods comprise binding to human or murine 4-1BB at a k _assoc of about 7.5×10 ⁵ 1/M·s or faster, at about 7.5×10 ⁵ 1 /M·s or faster k _assoc binds to human or murine 4-1BB at about 8 × 10 ⁵ l/M·s or faster k _assoc binds to human or murine 4-1BB at about 8.5 × 10 ⁵ 1/M·s or faster k _assoc binds to human or mouse 4-1BB, about 9 × 10 ⁵ 1/M·s or faster k _assoc binds to human or mouse 4-1BB , binding to human or mouse 4-1BB with a k _assoc of about 9.5 × 10 ⁵ 1/M·s or faster, or binding to human or mouse with a k _assoc of about 1 × 10 ⁶ 1/M·s or faster 4-1BB agonists of the class 4-1BB binding.

In some embodiments, the compositions, processes and methods described comprise binding to human or murine 4-1BB at a k _dissoc of about 2×10 ⁻⁵ 1/s or slower, and binding to human or murine 4-1BB at about 2.1×10 ⁻⁵ 1 /s or slower k _dissoc binds to human or murine 4-1BB, binds to human or murine 4-1BB at about 2.2 × 10 ^-5 1/s or slower k _dissoc , binds to human or murine 4-1BB at about 2.3 × 10 ^{- 5} 1/s or slower k _dissoc binds to human or murine 4-1BB, binds to human or murine 4-1BB at about 2.4 × 10 ^-5 1/s or slower k _dissoc , binds to human or murine 4-1BB at about 2.5 × 10 ^-5 1/s or slower k _dissoc to human or murine 4-1BB, about 2.6 × 10 ^-5 1/s or slower k _dissoc to human or murine 4-1BB, or Binds to human or murine 4-1BB at a k _dissoc of about 2.7 × 10 ^-5 1/s or slower, binds to human or murine 4-1BB at a k _dissoc of about 2.8 × 10 ^-5 1/s or slower , binding to human or murine 4-1BB at a k _dissoc of about 2.9 × 10 ^-5 1/s or slower, or binding to human or murine 4 at a k _dissoc of about 3 × 10 ^-5 1/s or slower - A 4-1BB agonist of 1BB binding.

In some embodiments, the compositions, processes and methods described include a 4-1BB agonist that binds to human or murine 4-1BB with an _IC50 of about 10 nM or less, Binds to human or murine 4-1BB with an IC ₅₀ of about 9 nM or less Binds to human or murine 4-1BB with an IC ₅₀ of about 8 nM or less Binds to human or murine 4-1BB with an IC ₅₀ of about 7 nM or less Binds to human or murine 4-1BB, binds to human or murine 4-1BB with an IC ₅₀ of about 6 nM or less, binds to human or murine 4-1BB with an IC ₅₀ of about 5 nM or less, Binds to human or murine 4-1BB with an IC ₅₀ of about 4 nM or less Binds to human or murine 4-1BB with an IC ₅₀ of about 3 nM or less Binds to human or murine 4-1BB with an IC ₅₀ of about 2 nM or less Binds to human or murine 4-1BB, or binds to human or murine 4-1BB with an IC ₅₀ of about 1 nM or less.

In some embodiments, the 4-1BB agonist is utumumab (also known as PF-05082566 or MOR-7480) or a fragment, derivative, variant or biosimilar thereof. Utumumab is commercially available from Pfizer, Inc. Utumumab is an immunoglobulin G2-λ anti-[ Homo sapiens TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T cell antigen ILA, CD137)] Homo sapiens (full human) single strain antibody. The amino acid sequence of utumumab is set forth in Table 6. Utumumab contains glycosylation sites at Asn59 and Asn292; at positions 22-96 (V _H -V _L ), 143-199 ( _CH 1-CL), 256-316 ( _CH 2) and 362-420 ( _CH 3 ) intrachain disulfide bonds in the heavy chain; disulfide bonds in the light chain at positions 22'-87' (V _H -V _L ) and 136'-195' ( _CH 1-CL) Sulfur bonds; at positions 218-218, 219-219, 222-222, and 225-225 of the IgG2A isotype, at positions 218-130, 219-219, 222-222, and 225-225 of the IgG2A/B isotype, and at positions of the IgG2B allotype interchain heavy chain-heavy chain disulfide bonds at positions 219-130(2), 222-222, and 225-225; 213' and 130-213' and the interchain heavy chain-light chain disulfide bond at positions 218-213' (2) of the IgG2B isotype. The preparation and properties of utumumab and its variants and fragments are described in U.S. Patent Nos. 8,821,867, 8,337,850, and 9,468,678 and International Patent Application Publication No. WO 2012/032433 A1, the disclosures of each of which are incorporated by reference way incorporated into this article. The preclinical characterization of utumumab is described in Fisher et al., Cancer Immunolog . & Immunother. 2012 , 61, 1721-33. Current clinical trials of utumumab in a variety of hematological and solid tumor indications include US National Institutes of Health (US National Institutes of Health) clinicaltrials.gov identification numbers NCT02444793, NCT01307267, NCT02315066 and NCT02554812.

In some embodiments, the 4-1BB agonist comprises a heavy chain provided by SEQ ID NO:42 and a light chain provided by SEQ ID NO:43. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively, or an antigen-binding fragment, a Fab fragment, a single chain variable chain thereof Fragment (scFv), variant or conjugate. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 99% identical to the sequences set forth in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 98% identical to the sequences set forth in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 97% identical to the sequences set forth in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequence set forth in SEQ ID NO:42 and SEQ ID NO:43, respectively.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of utumumab. In some embodiments, the 4-1BB agonist heavy chain variable region (V _H ) comprises the sequence set forth in SEQ ID NO: 44, and the 4-1BB agonist light chain variable region (V _L ) comprises The sequence shown in SEQ ID NO: 45, and conservative amino acid substitutions thereof. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 99% identical to the sequence set forth in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 98% identical to the sequence set forth in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 97% identical to the sequence set forth in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 96% identical to the sequence set forth in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 95% identical to the sequence set forth in SEQ ID NO:44 and SEQ ID NO:45, respectively. In some embodiments, the 4-1BB agonist comprises a scFv antibody comprising _VH and _VL regions that are each at least 99% identical to the sequences set forth in SEQ ID NO: 44 and SEQ ID NO: 45.

In some embodiments, the 4-1BB agonist comprises heavy chain CDR1, having the sequences set forth in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively, and conservative amino acid substitutions thereof, CDR2 and CDR3 domains; and light chain CDR1 , CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, respectively, and conservative amino acid substitutions.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by drug regulatory agencies with reference to utumumab. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody comprising at least 97% sequence identity, e.g., 97%, 98%, to the amino acid sequence of a reference drug or reference biological product , an amino acid sequence with 99% or 100% sequence identity, and it contains one or more post-translational modifications compared with the reference drug or reference biological product, wherein the reference drug or reference biological product is utumumab . In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application for authorization of a 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product , wherein the reference drug or reference biological product is utumumab. 4-1BB agonist antibodies can be authorized by drug regulatory agencies, such as US FDA and/or EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein The reference drug or reference biological product is utumumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein The reference drug or reference biological product is utumumab.

In some embodiments, the 4-1BB agonist is the monoclonal antibody usrelumab (also known as BMS-663513 and 20H4.9.h4a) or a fragment, derivative, variant or biosimilar thereof. Urelumab is commercially available from Bristol-Myers Squibb and Creative Biolabs, Inc. Urelumab is an immunoglobulin G4-κ anti-[ Homo sapiens TNFRSF9 (tumor necrosis factor receptor superfamily member 9, 4-1BB, T cell antigen ILA, CD137)] Homo sapiens (full human) monoclonal antibody. The amino acid sequence of Urelumab is set forth in Table 7. Urelumab contains an N-glycosylation site at position 298 (and 298''); at positions 22-95 (V _H -V _L ), 148-204 ( _CH 1-CL), 262- 322( _CH 2) and 368-426( _CH 3) (and at positions 22''-95'', 148''-204'', 262''-322'' and 368''-426'' ) heavy chain intrachain disulfide bonds; at positions 23'-88' (V _H -V _L ) and 136'-196' ( _CH 1-CL) (and at positions 23'''-88''' and 136'''-196''') light chain intrachain disulfide bonds; interchain heavy chain-heavy chain disulfide bonds at positions 227-227'' and 230-230''; and 135-216 ' and 135''-216''' interchain heavy chain-light chain disulfide bonds. The preparation and properties of usrelumab and variants and fragments thereof are described in US Patent Nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference. The preclinical and clinical features of Urelumab are described in Segal et al., Clin. Cancer Res. 2016 , available at http:/dx.doi.org/10.1158/1078-0432.CCR- 16-1272. Current clinical trials of Urelumab in various hematological and solid tumor indications include the National Institutes of Health clinicaltrials.gov identification numbers NCT01775631, NCT02110082, NCT02253992 and NCT01471210.

In some embodiments, the 4-1BB agonist comprises a heavy chain provided by SEQ ID NO:52 and a light chain provided by SEQ ID NO:53. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 52 and SEQ ID NO: 53, respectively, or an antigen-binding fragment, a Fab fragment, a single chain variable chain thereof Fragment (scFv), variant or conjugate. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 99% identical to the sequence set forth in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 98% identical to the sequences set forth in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 97% identical to the sequence set forth in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequence set forth in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequence set forth in SEQ ID NO:52 and SEQ ID NO:53, respectively.

In some embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of usrelumab. In some embodiments, the 4-1BB agonist heavy chain variable region (V _H ) comprises the sequence set forth in SEQ ID NO: 54 and the 4-1BB agonist light chain variable region (V _L ) comprises The sequence shown in SEQ ID NO: 55, and conservative amino acid substitutions thereof. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 99% identical to the sequence set forth in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 98% identical to the sequence set forth in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 97% identical to the sequence set forth in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 96% identical to the sequence set forth in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises _VH and _VL regions, each of which is at least 95% identical to the sequence set forth in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, the 4-1BB agonist comprises a scFv antibody comprising _VH and _VL regions that are each at least 99% identical to the sequences set forth in SEQ ID NO: 54 and SEQ ID NO: 55.

In some embodiments, the 4-1BB agonist comprises heavy chain CDR1, having the sequences set forth in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58 and conservative amino acid substitutions thereof, respectively. CDR2 and CDR3 domains; and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61, respectively, and conservative amino acid substitutions.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by the drug regulatory agency with reference to usrelumab. In some embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody comprising at least 97% sequence identity, e.g., 97%, 98%, to the amino acid sequence of a reference drug or reference biological product. Amino acid sequences with %, 99% or 100% sequence identity and which contain one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is usrelumab . In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or application for authorization of a 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product , wherein the reference drug or reference biological product is Urelumab. 4-1BB agonist antibodies can be authorized by drug regulatory agencies, such as US FDA and/or EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is Urelumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is Urelumab.

In some embodiments, the 4-1BB agonist is selected from the group consisting of 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (Leinco Technologies B591), the antibody produced by the cell line deposited as ATCC No. HB-11248 and disclosed in US Patent No. 6,974,863, 5F4 (BioLegend 31 1503), C65-485 (BD Pharmingen 559446), US Patent Application Antibodies disclosed in Publication No. US 2005/0095244, antibodies disclosed in U.S. Patent No. 7,288,638 (such as 20H4.9-IgG1 (BMS-663031 )), antibodies disclosed in U.S. Patent No. 6,887,673 (such as 4E9 or BMS -554271), the antibodies disclosed in U.S. Patent No. 7,214,493, the antibodies disclosed in U.S. Patent No. 6,303,121, the antibodies disclosed in U.S. Patent No. 6,569,997, the antibodies disclosed in U.S. Patent No. 6,905,685 (such as 4E9 or BMS-554271 ), antibodies disclosed in U.S. Patent No. 6,362,325 (such as 1D8 or BMS-469492; 3H3 or BMS-469497; or 3El), antibodies disclosed in U.S. Patent No. 6,974,863 (such as 53A2); U.S. Patent No. 6,210,669 Antibodies (such as 1D8, 3B8 or 3El), antibodies described in U.S. Patent No. 5,928,893, antibodies disclosed in U.S. Patent No. 6,303,121, antibodies disclosed in U.S. Patent No. 6,569,997, International Patent Application Publication No. WO 2012/ 177788, the antibodies disclosed in WO 2015/119923 and WO 2010/042433, and fragments, derivatives, conjugates, variants or biosimilars thereof, the disclosure of each of the foregoing patents or patent application publications Incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist fusion protein described in: International Patent Application Publication No. WO 2008/025516 A1, WO 2009/007120 A1, WO 2010/ 003766 A1, WO 2010/010051 A1 and WO 2010/078966 A1; US Patent Application Publication Nos. US 2011/0027218 A1, US 2015/0126709 A1, US 2011/0111494 A1, US 2015/0110734 A1 and US 2015/0126710 A1; and US Patent Nos. 9,359,420, 9,340,599, 8,921,519 and 8,450,460, the disclosures of which are incorporated herein by reference.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist fusion protein as depicted in Structure IA (C-terminal Fc antibody fragment fusion protein) or Structure IB (N-terminal Fc antibody fragment fusion protein), or Fragments, derivatives, conjugates, variants or biosimilars thereof (see Figure 18). In structures IA and IB, cylinders refer to individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF binding domains derived from, for example, 4-1BBL (4-1BB ligand, CD137 ligand (CD137L) or tumor necrosis factor superfamily member 9 (TNFSF9)) Or binding to 4-1BB antibodies, the TNFRSF binding domains fold to form a trivalent protein, which is then linked to two trivalent proteins via IgG1-Fc (including CH3 and CH2 domains), followed by disulfide bonds (elongated Small oval) links the two trivalent proteins together, stabilizing the structure and providing an agonist capable of bringing together the intracellular signaling domains of the six receptors and signaling the proteins to form a signaling complex. A TNFRSF binding domain represented as a cylinder may be a scFv domain comprising, for example, _VH and _VL chains linked by a linker, which may comprise hydrophilic residues and Gly and Ser sequences that provide flexibility and provide solubility. Glu and Lys. Any scFv domain design can be used, such as those described in de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad et al., Clin. & Dev. Immunol.), 2012, 980250; Monnier et al., Antibodies, 2013, 2, 193-208; or references incorporated elsewhere herein. Fusion protein structures of this form are described in US Patent Nos. 9,359,420, 9,340,599, 8,921,519 and 8,450,460, the disclosures of which are incorporated herein by reference.

The amino acid sequences of the other polypeptide domains of Structure IA provided in Figure 18 can be found in Table 8. The Fc domain preferably comprises an entire constant domain (amino acids 17-230 of SEQ ID NO:62), an entire hinge domain (amino acids 1-16 of SEQ ID NO:62) or a portion of a hinge domain (e.g., SEQ ID NO: :62 amino acids 4-16). Preferred linkers for linking C-terminal Fc antibodies can be selected from the examples provided in SEQ ID NO:63 to SEQ ID NO:72, including linkers suitable for fusion with other polypeptides.

The amino acid sequences of the other polypeptide domains of Structure IB provided in Figure 18 can be found in Table 9. If the Fc antibody fragment is fused with the N-terminal of the TNRFSF fusion protein as in structure IB, the sequence of the Fc module is preferably the sequence shown in SEQ ID NO: 73, and the linker sequence is preferably selected from SED ID NO :74 to the embodiment shown in SEQ ID NO:76.

In some embodiments, the 4-1BB agonist fusion protein according to Structure I-A or Structure I-B comprises one or more 4-1BB binding domains selected from the group consisting of: variable heavy chain of utumumab and a variable light chain, a variable heavy chain and a variable light chain of Urelumab, a variable heavy chain and a variable light chain of Urelumab, a variable heavy chain and a variable light chain selected from those described in Table 10 and Variable heavy chains and variable light chains of variable light chains, any combination of the aforementioned variable heavy chains and variable light chains, and fragments, derivatives, conjugates, variants and biosimilars thereof.

In some embodiments, a 4-1BB agonist fusion protein according to Structure I-A or I-B comprises one or more 4-1BB binding domains comprising a 4-1BBL sequence. In some embodiments, the 4-1BB agonist fusion protein according to Structure I-A or Structure I-B comprises one or more 4-1BB binding domains comprising the sequence according to SEQ ID NO:77. In some embodiments, a 4-1BB agonist fusion protein according to Structure I-A or I-B comprises one or more 4-1BB binding domains comprising a soluble 4-1BBL sequence. In some embodiments, the 4-1BB agonist fusion protein according to Structure I-A or Structure I-B comprises one or more 4-1BB binding domains comprising the sequence according to SEQ ID NO:78.

In some embodiments, a 4-1BB agonist fusion protein according to Structure IA or Structure IB comprises one or more 4-1BB binding domains comprising each of SEQ ID NO: 43 and SEQ ID NO : scFv domains of _VH and _VL regions with at least 95% identity to the sequences shown in 44, wherein the _VH and _VL domains are connected via a linker. In some embodiments, a 4-1BB agonist fusion protein according to Structure IA or Structure IB comprises one or more 4-1BB binding domains comprising each of SEQ ID NO: 54 and SEQ ID NO : scFv domains of _VH and _VL regions with at least 95% identity to the sequences shown in 55, wherein the _VH and _VL domains are connected via a linker. In some embodiments, a 4-1BB agonist fusion protein according to Structure IA or IB includes one or more 4-1BB binding domains comprising _VH and _VL , respectively, as set forth in Table 10 scFv domains of _VH and _VL regions with at least 95% identity in sequence, wherein the _VH and _VL domains are connected via a linker.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist single chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) The second soluble 4-1BB binding domain, (iv) the second peptide linker, and (v) the third soluble 4-1BB binding domain further comprise an additional domain at the N-terminus and/or C-terminus, and wherein the additional domain It is a Fab or Fc fragment domain. In some embodiments, the 4-1BB agonist is a 4-1BB agonist single chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) The second soluble 4-1BB binding domain, (iv) the second peptide linker, and (v) the third soluble 4-1BB binding domain further comprise an additional domain at the N-terminus and/or C-terminus, and wherein the additional domain is a Fab or Fc fragment domain, wherein each of the soluble 4-1BB domains lacks a stem region (which facilitates trimerization and provides some distance from the cell membrane, but is not part of the 4-1BB binding domain) and the first peptide The linker and the second peptide linker independently have a length of 3-8 amino acids.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist single-chain fusion polypeptide comprising (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide Linker, (iii) the second soluble TNF superfamily cytokine domain, (iv) the second peptide linker, and (v) the third soluble TNF superfamily cytokine domain, wherein the soluble TNF superfamily cytokine domain Each of these lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein each TNF superfamily interleukin domain is a 4-1BB binding domain.

In some embodiments, the 4-1BB agonist is a 4-1BB agonist scFv antibody comprising any of the foregoing _VH domains linked to any of the foregoing _VL domains.

In some embodiments, the 4-1BB agonist is BPS Bioscience 4-1BB Agonist Antibody, Cat. No. 79097-2, commercially available from BPS Bioscience, San Diego, CA (BPS Bioscience, San Diego, CA, USA) . In some embodiments, the 4-1BB agonist is Creative Biolabs 4-1BB agonist antibody, catalog number MOM-18179, available from Creative Biolabs, Shirley, NY, USA ). 3. OX40(CD134) agonists

In some embodiments, the TNFRSF agonist is an OX40 (CD134) agonist. The OX40 agonist can be any OX40 binding molecule known in the art. The OX40-binding molecule can be a monoclonal antibody or a fusion protein capable of binding to human or mammalian OX40. An OX40 agonist or an OX40 binding molecule may comprise any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of an immunoglobulin molecule Immunoglobulin heavy chain. An OX40 agonist or OX40 binding molecule can have a heavy chain and a light chain. As used herein, the term binding molecule also includes antibodies (including full-length antibodies), monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), human antibodies, humanized or Chimeric antibodies and antibody fragments, such as Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the foregoing and engineered versions of antibodies that bind OX40, such as scFv molecules. In some embodiments, the OX40 agonist is an antigen binding protein of a fully human antibody. In some embodiments, the OX40 agonist is an antigen binding protein of a humanized antibody. In some embodiments, OX40 agonists for use in the methods and compositions of the present disclosure include anti-OX40 antibodies, human anti-OX40 antibodies, mouse anti-OX40 antibodies, mammalian anti-OX40 antibodies, monoclonal anti-OX40 antibodies, polyclonal Anti-OX40 antibody, chimeric anti-OX40 antibody, anti-OX40 adnectin, anti-OX40 domain antibody, single chain anti-OX40 fragment, heavy chain anti-OX40 fragment, light chain anti-OX40 fragment, anti-OX40 fusion protein, and Fragments, derivatives, conjugates, variants or biosimilars. In some embodiments, the OX40 agonist is an agonistic anti-OX40 humanized or fully human monoclonal antibody (ie, an antibody derived from a single cell line).

In some embodiments, the OX40 agonist or OX40 binding molecule can also be a fusion protein. OX40 fusion proteins comprising an Fc domain fused to OX40L are described eg in Sadun et al., J Immunotherapeutics 2009, 182, 1481-89. In some embodiments, multimeric OX40 agonists, such as trimeric or hexameric OX40 agonists (with three or six ligand body-binding domain) induces clustering of elite receptors (OX40L) and formation of internal cell-messaging complexes. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins comprising three TNFRSF binding domains and IgG1-Fc, optionally further linking two or more of these fusion proteins are described, for example, in Gieffers et al. People, Molecular Cancer Therapeutics 2013, 12, 2735-47.

Potent OX40 antibodies and fusion proteins are known to induce strong immune responses. Curti et al., Cancer Research 2013, 73, 7189-98. In some embodiments, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to the OX40 antigen in a manner sufficient to reduce toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that abrogates antibody-dependent cellular cytotoxicity (ADCC), such as NK cytotoxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that abolishes antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that abolishes complement-dependent cytotoxicity (CDC). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that abolishes Fc region function.

In some embodiments, the OX40 agonist is characterized as binding to human OX40 (SEQ ID NO: 85) with high affinity and agonistic activity. In some embodiments, the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO: 85). In some embodiments, the OX40 agonist is a binding molecule that binds to murine OX40 (SEQ ID NO: 86). Amino acid sequences of OX40 antigens that bind to OX40 agonists or binding molecules are summarized in Table 11.

In some embodiments, the described compositions, procedures and methods include an OX40 agonist that binds human or murine OX40 with a KD of about 100 pM or less, with a KD of about 90 pM or less Binds human or murine OX40, binds human or murine OX40 with a KD of about 80 pM or less, binds human or murine OX40 with a KD of about 70 pM or less, binds human with a KD of about 60 pM or less or murine OX40, binds human or murine OX40 with a KD of about 50 pM or less, binds human or murine OX40 with a KD of about 40 pM or less, or binds human or murine OX40 with a KD of about 30 pM or less Class OX40.

In some embodiments, the described compositions, processes and methods include an OX40 agonist that interacts with human or murine OX40 at a k _assoc of about 7.5 x ¹⁰⁵ 1/M·s or faster Binding, binding to human or mouse OX40 at a k _assoc of about 7.5 × 10 ⁵ 1/M·s or faster, binding to human or mouse OX40 at a k _assoc of about 8 × 10 ⁵ 1/M·s or faster Binding, binding to human or mouse OX40 at a k _assoc of about 8.5 × 10 ⁵ 1/M·s or faster, binding to human or mouse OX40 at a k _assoc of about 9 × 10 ⁵ 1/M·s or faster Binds to human or murine OX40 at a k _assoc of about 9.5 × 10 ⁵ 1/M·s or faster or to human or murine OX40 at a k _assoc of about 1 × 10 ⁶ 1/M·s or faster combined.

In some embodiments, the described compositions, processes and methods include an OX40 agonist that binds to human or murine OX40 with a k _dissoc of about 2 x 10 ^-5 1/s or slower , binding to human or mouse OX40 with a k _dissoc of about 2.1 × 10 ^-5 1/s or slower, binding to human or mouse OX40 with a k _dissoc of about 2.2 × 10 ^-5 1/s or slower, and About 2.3 × 10 ^-5 1/s or slower k _dissoc binds to human or murine OX40, about 2.4 × 10 ^-5 1/s or slower k _dissoc binds to human or murine OX40, about 2.5 × 10 ^-5 1/s or slower k _dissoc binds to human or mouse OX40, binds to human or mouse OX40 at about 2.6 × 10 ^-5 1/s or slower k _dissoc , binds to human or mouse OX40 at about 2.7 × 10 ^-5 1/s or slower k _dissoc binds to human or mouse OX40, about 2.8 × 10 ^-5 1/s or slower k _dissoc binds to human or mouse OX40, about 2.9 × 10 ^-5 Binds to human or murine OX40 at a k _dissoc of 1/s or slower or binds to human or murine OX40 at a k _dissoc of about 3 × 10 ^-5 1/s or slower.

In some embodiments, the described compositions, processes and methods include an OX40 agonist that binds to human or murine _OX40 with an IC50 of about 10 nM or less, at about 9 nM or Binds to human or murine OX40 with a lower _IC50 , binds to human or murine _OX40 with an IC50 of about 8 nM or less, binds to human or murine OX40 with an _IC50 of about 7 nM or less, and Binds human or murine OX40 with an IC ₅₀ of about 6 nM or less, binds human or murine OX40 with an IC ₅₀ of about 5 nM or less, binds human or murine OX40 with an IC ₅₀ of about 4 nM or less OX40 binds, binds to human or murine _OX40 with an IC 50 of about 3 nM or less, binds to human or murine OX40 with an IC ₅₀ of about 2 nM or less, or binds to human or murine OX40 with an IC ₅₀ of about 1 nM or less Human or murine OX40 binding.

In some embodiments, the OX40 agonist is tavoximab, also known as MEDI0562 or MEDI-0562. Tavocitumab is available from the MedImmune subsidiary of AstraZeneca, Inc. Tavoximab is an immunoglobulin G1-κ anti-[Homo sapiens TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134)] humanized and chimeric monoclonal antibody. The amino acid sequence of tavoximab is set forth in Table 12. Tavocizumab contains N-glycosylation sites at positions 301 and 301'', with fucosylation complex biantennary CHO-type glycans; at positions 22-95 (V _H -V _L ), 148-204 (( _CH 1- _CL ), 265-325 ( _CH 2) and 371-429 ( _CH 3) (and at positions 22''-95'', 148''-204'' , 265''-325'' and 371''-429'') heavy chain intrachain disulfide bonds; at positions 23'-88'(V _H -V _L ) and 134'-194'(C _H 1- _CL ) (and at positions 23'''-88''' and 134'''-194'''); at positions 230-230'' and 233- interchain heavy chain-heavy chain disulfide bond at 233''; and interchain heavy chain-light chain disulfide bond at 224-214' and 224''-214'''. Current clinical trials in solid tumor indications include National Institutes of Health clinicaltrials.gov identifiers NCT02318394 and NCT02705482.

In some embodiments, the OX40 agonist comprises a heavy chain provided by SEQ ID NO:87 and a light chain provided by SEQ ID NO:88. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 87 and SEQ ID NO: 88, respectively, or an antigen-binding fragment, a Fab fragment, a single chain variable fragment thereof ( scFv), variants or combinations. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 99% identical to the sequences set forth in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 98% identical to the sequences set forth in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 97% identical to the sequence set forth in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequence set forth in SEQ ID NO:87 and SEQ ID NO:88, respectively.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of tavoximab. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence set forth in SEQ ID NO:89 and the OX40 agonist light chain variable region ( _VL ) comprises SEQ ID NO:90 The sequence shown in , and its conservative amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 99% identical to the sequence set forth in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 98% identical to the sequence set forth in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 97% identical to the sequence set forth in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 96% identical to the sequence set forth in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 95% identical to the sequence set forth in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, the OX40 agonist comprises a scFv antibody comprising _VH and _VL regions that are each at least 99% identical to the sequences set forth in SEQ ID NO:89 and SEQ ID NO:90, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR1 having the sequences shown in SEQ ID NO:91, SEQ ID NO:92, and SEQ ID NO:93, respectively, and conservative amino acid substitutions thereof. CDR3 domains; and light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO:94, SEQ ID NO:95 and SEQ ID NO:96, respectively, and conservative amino acid substitutions thereof.

In some embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by drug regulatory agencies with reference to tavoximab. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, 99%, or An amino acid sequence with 100% sequence identity and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is tavoximab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an OX40 agonist antibody licensed or applying for authorization, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference The drug or reference biological product is tavoximab. OX40 agonist antibodies can be authorized by drug regulatory agencies, such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is tavoximab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is tavoximab.

In some embodiments, the OX40 agonist is 11D4, which is a fully human antibody available from Pfizer, Inc. The preparation and characterization of 11D4 is described in US Patent Nos. 7,960,515, 8,236,930 and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 11D4 is set forth in Table 13.

In some embodiments, the OX40 agonist comprises a heavy chain provided by SEQ ID NO:97 and a light chain provided by SEQ ID NO:98. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 97 and SEQ ID NO: 98, respectively, or an antigen-binding fragment, a Fab fragment, a single chain variable fragment thereof ( scFv), variants or combinations. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 99% identical to the sequences set forth in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 98% identical to the sequences set forth in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 97% identical to the sequences set forth in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NO:97 and SEQ ID NO:98, respectively.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 11D4. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence set forth in SEQ ID NO:99 and the OX40 agonist light chain variable region ( _VL ) comprises SEQ ID NO:100 The sequence shown in , and its conservative amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 99% identical to the sequences set forth in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 98% identical to the sequences set forth in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 97% identical to the sequences set forth in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 96% identical to the sequences set forth in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 95% identical to the sequences set forth in SEQ ID NO:99 and SEQ ID NO:100, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR1 having the sequences set forth in SEQ ID NO: 101, SEQ ID NO: 102, and SEQ ID NO: 103, respectively, and conservative amino acid substitutions thereof. CDR3 domains; and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, respectively, and conservative amino acid substitutions.

In some embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by Drug Regulatory Agency reference 11D4. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, 99%, or An amino acid sequence with 100% sequence identity and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is 11D4. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an OX40 agonist antibody licensed or applying for authorization, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference Drug or reference biological product is 11D4. OX40 agonist antibodies can be authorized by drug regulatory agencies, such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the Reference drug or reference biological product is 11D4. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the Reference drug or reference biological product is 11D4.

In some embodiments, the OX40 agonist is 18D8, a fully human antibody available from Pfizer, Inc. The preparation and characterization of 18D8 is described in US Patent Nos. 7,960,515, 8,236,930 and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 18D8 is set forth in Table 14.

In some embodiments, the OX40 agonist comprises the heavy chain provided by SEQ ID NO:107 and the light chain provided by SEQ ID NO:108. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 107 and SEQ ID NO: 108, respectively, or antigen-binding fragments, Fab fragments, single chain variable fragments thereof ( scFv), variants or combinations. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 99% identical to the sequences set forth in SEQ ID NO: 107 and SEQ ID NO: 108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 98% identical to the sequences set forth in SEQ ID NO: 107 and SEQ ID NO: 108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 97% identical to the sequences set forth in SEQ ID NO: 107 and SEQ ID NO: 108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO: 107 and SEQ ID NO: 108, respectively. In some embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NO: 107 and SEQ ID NO: 108, respectively.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 18D8. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence set forth in SEQ ID NO: 109 and the OX40 agonist light chain variable region ( _VL ) comprises SEQ ID NO: 110 The sequence shown in , and its conservative amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 99% identical to the sequences set forth in SEQ ID NO: 109 and SEQ ID NO: 110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 98% identical to the sequences set forth in SEQ ID NO: 109 and SEQ ID NO: 110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 97% identical to the sequences set forth in SEQ ID NO: 109 and SEQ ID NO: 110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 96% identical to the sequences set forth in SEQ ID NO: 109 and SEQ ID NO: 110, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 95% identical to the sequences set forth in SEQ ID NO: 109 and SEQ ID NO: 110, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR1 having the sequences set forth in SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113, respectively, and conservative amino acid substitutions thereof. CDR3 domains; and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116, respectively, and conservative amino acid substitutions.

In some embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the Drug Regulatory Agency with reference to 18D8. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, 99%, or An amino acid sequence with 100% sequence identity and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is 18D8. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an OX40 agonist antibody licensed or applying for authorization, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference Drug or reference biological product is 18D8. OX40 agonist antibodies can be authorized by drug regulatory agencies, such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the Reference drug or reference biological product is 18D8. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the Reference drug or reference biological product is 18D8.

In some embodiments, the OX40 agonist is Hu119-122, which is a humanized antibody available from GlaxoSmithKline plc. The preparation and characterization of Hu119-122 are described in US Patent Nos. 9,006,399 and 9,163,085 and International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu119-122 is set forth in Table 15.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu119-122. In some embodiments, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence set forth in SEQ ID NO: 117 and the OX40 agonist light chain variable region ( _VL ) comprises SEQ ID NO: 118 The sequence shown in , and its conservative amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 99% identical to the sequences set forth in SEQ ID NO: 117 and SEQ ID NO: 118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 98% identical to the sequences set forth in SEQ ID NO: 117 and SEQ ID NO: 118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 97% identical to the sequences set forth in SEQ ID NO: 117 and SEQ ID NO: 118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 96% identical to the sequences set forth in SEQ ID NO: 117 and SEQ ID NO: 118, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 95% identical to the sequences set forth in SEQ ID NO: 117 and SEQ ID NO: 118, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR1 having the sequences set forth in SEQ ID NO: 119, SEQ ID NO: 120, and SEQ ID NO: 121, respectively, and conservative amino acid substitutions thereof. CDR3 domains; and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 122, SEQ ID NO: 123 and SEQ ID NO: 124, respectively, and conservative amino acid substitutions.

In some embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the Drug Administration with reference to Hu119-122. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, 99%, or An amino acid sequence with 100% sequence identity and comprising one or more post-translational modifications compared with the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu119-122. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an OX40 agonist antibody licensed or applying for authorization, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference The drug or reference biological product is Hu119-122. OX40 agonist antibodies can be authorized by drug regulatory agencies, such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is Hu119-122. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is Hu119-122.

In some embodiments, the OX40 agonist is Hu106-222, a humanized antibody available from GlaxoSmithKline plc. The preparation and characterization of Hu106-222 are described in US Patent Nos. 9,006,399 and 9,163,085 and International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu106-222 is set forth in Table 16.

In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu106-222. In some embodiments, the OX40 agonist heavy chain variable region (V _H ) comprises the sequence set forth in SEQ ID NO: 125, and the OX40 agonist light chain variable region (V _L ) comprises SEQ ID NO: 126 The sequence shown in , and its conservative amino acid substitutions. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 99% identical to the sequence set forth in SEQ ID NO: 125 and SEQ ID NO: 126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 98% identical to the sequence set forth in SEQ ID NO: 125 and SEQ ID NO: 126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 97% identical to the sequences set forth in SEQ ID NO: 125 and SEQ ID NO: 126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 96% identical to the sequence set forth in SEQ ID NO: 125 and SEQ ID NO: 126, respectively. In some embodiments, the OX40 agonist comprises _VH and _VL regions that are each at least 95% identical to the sequence set forth in SEQ ID NO: 125 and SEQ ID NO: 126, respectively.

In some embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR1 having the sequences set forth in SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively, and conservative amino acid substitutions thereof. a CDR3 domain; and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132, respectively, and conservative amino acid substitutions.

In some embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the Drug Administration with reference to Hu106-222. In some embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, 99%, or An amino acid sequence with 100% sequence identity and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu106-222. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an OX40 agonist antibody licensed or applying for authorization, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference The drug or reference biological product is Hu106-222. OX40 agonist antibodies can be authorized by drug regulatory agencies, such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference medicine or reference biological product is Hu106-222. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference medicine or reference biological product is Hu106-222.

In some embodiments, the OX40 agonist antibody is MEDI6469 (also known as 9B12). MEDI6469 is a murine monoclonal antibody. Weinberg et al., Journal of Immunotherapy 2006, 29 , 575-585 . In some embodiments, the OX40 agonist is an antibody produced by the 9B12 hybridoma and deposited with Biovest Inc. (Malvern, MA, USA) as described in Weinberg et al., Journal of Immunotherapy 2006 , 29, 575-585, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the antibody comprises the CDR sequences of MEDI6469. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of MEDI6469.

In some embodiments, the OX40 agonist is L106 BD (Pharmingen, Product No. 340420). In some embodiments, the OX40 agonist comprises the CDRs of antibody L106 (BD Pharmingen, Product No. 340420). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody L106 (BD Pharmingen, Product No. 340420). In some embodiments, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, Cat. No. 20073). In some embodiments, the OX40 agonist comprises the CDRs of the antibody ACT35 (Santa Cruz Biotechnology, catalog #20073). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, catalog number 20073). In some embodiments, the OX40 agonist is a murine monoclonal antibody anti-mCD134/mOX40 (clone OX86), available as InVivoMAb from BioXcell Inc, West Lebanon, NH.

In some embodiments, the OX40 agonist is selected from the OX40 agonists described in International Patent Application Publication Nos. WO 95/12673, WO 95/21925, WO 2006/121810, WO 2012/027328, WO 2013/028231, WO 2013/038191 and WO 2014/148895; European Patent Application EP 0672141; US Patent Application Publication Nos. US 2010/136030, US 2014/377284 No., US 2015/190506 and US 2015/132288 (including pure lines 20E5 and 12H3); Nos. 8,133,983, 9,006,399, and 9,163,085, the disclosures of each of which are incorporated herein by reference in their entirety.

In some embodiments, the OX40 agonist is an OX40 agonist fusion protein as depicted in Structure I-A (C-terminal Fc-antibody fragment fusion protein) or Structure I-B (N-terminal Fc-antibody fragment fusion protein), or Fragments, derivatives, conjugates, variants or biosimilars. The properties of Structures I-A and I-B are described above and in US Patent Nos. 9,359,420, 9,340,599, 8,921,519 and 8,450,460, the disclosures of which are incorporated herein by reference. The amino acid sequence of the polypeptide domain of Structure I-A provided in Figure 18 can be found in Table 9. The Fc domain preferably comprises an entire constant domain (amino acids 17-230 of SEQ ID NO:62), an entire hinge domain (amino acids 1-16 of SEQ ID NO:62) or a portion of a hinge domain (e.g., SEQ ID NO: :62 amino acids 4-16). Preferred linkers for linking C-terminal Fc antibodies can be selected from the examples provided in SEQ ID NO:63 to SEQ ID NO:72, including linkers suitable for fusion with other polypeptides. Similarly, the amino acid sequences of the polypeptide domains of Structure I-B provided in Figure 18 can be found in Table 10. If the Fc antibody fragment is fused with the N-terminal of the TNRFSF fusion protein as in structure I-B, the sequence of the Fc module is preferably the sequence shown in SEQ ID NO: 73, and the linker sequence is preferably selected from SED ID NO :74 to the embodiment shown in SEQ ID NO:76.

In some embodiments, the OX40 agonist fusion protein according to Structure I-A or Structure I-B comprises one or more OX40 binding domains selected from the group consisting of: the variable heavy chain and the variable light chain of tavoximab , the variable heavy chain and variable light chain of 11D4, the variable heavy chain and variable light chain of 18D8, the variable heavy chain and variable light chain of Hu119-122, the variable heavy chain and variable light chain of Hu106-222 Light chains, variable heavy chains and variable light chains selected from the variable heavy chains and variable light chains described in Table 17, any combination of the aforementioned variable heavy chains and variable light chains, and fragments, derivatives thereof substances, conjugates, variants and biosimilars.

In some embodiments, an OX40 agonist fusion protein according to Structure I-A or I-B comprises one or more OX40 binding domains comprising an OX40L sequence. In some embodiments, the OX40 agonist fusion protein according to Structure I-A or Structure I-B comprises one or more OX40 binding domains comprising the sequence according to SEQ ID NO:133. In some embodiments, an OX40 agonist fusion protein according to Structure I-A or I-B comprises one or more OX40 binding domains comprising a soluble OX40L sequence. In some embodiments, the OX40 agonist fusion protein according to Structure I-A or Structure I-B comprises one or more OX40 binding domains comprising the sequence according to SEQ ID NO:134. In some embodiments, the OX40 agonist fusion protein according to Structure I-A or Structure I-B comprises one or more OX40 binding domains comprising a sequence according to SEQ ID NO:135.

In some embodiments, the OX40 agonist fusion protein according to Structure IA or Structure IB comprises one or more OX40 binding domains, the one or more binding domains are scFv domains comprising each of SEQ ID NO: 89 and a _VH and _VL region at least 95% identical to the sequence shown in SEQ ID NO: 90, wherein the _VH and _VL domains are connected by a linker. In some embodiments, the OX40 agonist fusion protein according to Structure IA or Structure IB comprises one or more OX40 binding domains, the one or more binding domains are scFv domains comprising each of SEQ ID NO: 99 and a _VH and _VL region at least 95% identical to the sequence shown in SEQ ID NO: 100, wherein the _VH and _VL domains are connected by a linker. In some embodiments, the OX40 agonist fusion protein according to Structure IA or Structure IB comprises one or more OX40 binding domains, the one or more binding domains are scFv domains comprising each of SEQ ID NO: 109 and a _VH and _VL region at least 95% identical to the sequence shown in SEQ ID NO: 110, wherein the _VH and _VL domains are connected by a linker. In some embodiments, the OX40 agonist fusion protein according to Structure IA or Structure IB comprises one or more OX40 binding domains, the one or more binding domains are scFv domains comprising each of SEQ ID NO: 127 and a _VH and _VL region at least 95% identical to the sequence shown in SEQ ID NO: 128, wherein the _VH and _VL domains are connected by a linker. In some embodiments, the OX40 agonist fusion protein according to Structure IA or Structure IB comprises one or more OX40 binding domains, the one or more binding domains are scFv domains comprising each of SEQ ID NO: 125 and a _VH and _VL region at least 95% identical to the sequence shown in SEQ ID NO: 126, wherein the _VH and _VL domains are connected by a linker. In some embodiments, an OX40 agonist fusion protein according to Structure IA or IB comprises one or more OX40 binding domains that are scFv domains comprising each of the compounds provided in Table 17 A _VH and _VL region in which the _VH and _VL sequences are at least 95% identical, wherein the _VH and _VL domains are connected by a linker.

In some embodiments, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising: (i) a first soluble OX40 binding domain; (ii) a first peptide linker; (iii) a second soluble OX40 binding domain; (iv) a second peptide linker; and (v) a third soluble OX40 binding domain further comprising an additional domain at the N-terminus and/or C-terminus, and wherein the additional domain is a Fab or Fc fragment domain. In some embodiments, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising: (i) a first soluble OX40 binding domain; (ii) a first peptide linker; (iii) a second soluble OX40 binding domain; (iv) a second peptide linker; and (v) a third soluble OX40 binding domain, which further comprises an additional domain at the N-terminus and/or C-terminus, wherein the additional domain is a Fab or Fc fragment domain, wherein Each of the soluble OX40-binding domains lacks a stem region (which facilitates trimerization and provides some distance from the cell membrane, but is not part of the OX40-binding domain) and the first and second peptide linkers independently have a 3- 8 amino acids in length.

In some embodiments, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising: (i) a first soluble tumor necrosis factor (TNF) superfamily interleukin domain; (ii) a first peptide linker (iii) the second soluble TNF superfamily cytokine domain; (iv) the second peptide linker; and (v) the third soluble TNF superfamily cytokine domain, wherein the soluble TNF superfamily cytokine domain Each lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein the TNF superfamily interleukin domain is an OX40 binding domain.

In some embodiments, the OX40 agonist is MEDI6383. MEDI6383 is an OX40 agonistic fusion protein and can be prepared as described in US Patent No. 6,312,700, the disclosure of which is incorporated herein by reference.

In some embodiments, the OX40 agonist is an OX40 agonist scFv antibody comprising any of the foregoing _VH domains linked to any of the foregoing _VL domains.

In some embodiments, the OX40 agonist is Creative Biolabs OX40 agonist monoclonal antibody MOM-18455, available from Creative Biolabs, Inc., Shirley, New York, USA.

In some embodiments, the OX40 agonist is the OX40 agonist antibody clone Ber-ACT35, commercially available from BioLegend, Inc., San Diego, CA, USA. C. Optional cell viability analysis

Optionally, following an initial first expansion (sometimes referred to as initial bulk expansion), cell viability assays can be performed using standard assays known in the art. Accordingly, in certain embodiments, the method comprises performing a cell viability assay after initiating the first expansion. For example, a trypan blue exclusion assay, which selectively marks dead cells and allows assessment of viability, can be performed on bulk TIL samples. Other assays for testing viability may include, but are not limited to, Alamar blue assays and MTT assays. 1. Cell Counting, Viability, Flow Cytometry

In some embodiments, cell count and/or viability are measured. Expression of markers such as, but not limited to, CD3, CD4, CD8, and CD56 and any other marker disclosed or described herein can be measured by flow cytometry using a FACSCanto ^™ flow cytometer (BD Biosciences )), measured with antibodies such as, but not limited to, those commercially available from Biosciences (Biosciences, San Jose, CA). Cells can be counted manually using a disposable c-chip hemocytometer (VWR, Batavia, IL) and viability can be assessed using any method known in the art, including but not limited to trypan blue staining. Cell viability can also be analyzed based on US Patent Application Publication No. 2018/0282694, which is incorporated herein by reference in its entirety. Cell viability can also be analyzed based on US Patent Application Publication No. 2018/0280436 or International Patent Application Publication No. WO/2018/081473, both of which are incorporated herein in their entirety for all purposes.

In some cases, the subject TIL population can be cryopreserved immediately using the protocols discussed below. Alternatively, the subject TIL population can be REP and then cryopreserved as discussed below. Similarly, in cases where genetically modified TILs are to be used in therapy, a subject or population of REP TILs can be genetically modified for appropriate therapy. 2. Cell culture

In some embodiments, methods for amplifying TILs (including those discussed above and in FIGS. 1 and 8 (especially, for example, FIGS. 8A and/or 8E and/or the method exemplified in FIG. 8F and/or FIG. 8G)) may include using about 5,000 mL to about 25,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, the medium is serum-free medium. In some embodiments, the medium in the initial first expansion is serum-free. In some embodiments, the medium in the second expansion is serum-free. In some embodiments, the medium in both the initial first expansion and the second expansion (also referred to as rapid second expansion) is serum-free. In some embodiments, no more than one type of cell culture medium is used to expand the number of TILs. Any suitable cell culture medium can be used, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamycin sulfate) cell culture medium (Invitrogen, Calif. Carlsbad CA). In this regard, the methods of the invention advantageously reduce the amount of media and the number of media types required to expand the number of TILs. In some embodiments, expanding TIL numbers can comprise feeding the cells no more frequently than once every three or four days. Expansion of cell numbers in gas-permeable vessels Simplifies the procedures required to expand cell numbers by reducing the frequency of feeding required to expand cells.

In some embodiments, the cell culture medium in the first and/or second gas permeable container is unfiltered. The use of unfiltered cell culture media simplifies the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and/or second gas permeable container lacks beta-mercaptoethanol (BME).

In some embodiments, the method comprises obtaining a tumor tissue sample from a mammal during the method; culturing the tumor tissue sample in a first gas-permeable container for a duration of about 1 to 8 days, e.g., about 7 days to initiate the first expansion Or cultivate for about 8 days as the initial expansion of the first gas permeable container containing cell culture medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3; transfer the TILs to a second gas permeable container and Expanding the number of TILs in a gas-permeable container for about 7 to 9 days, such as about 7 days, about 8 days, or about 9 days, the second gas-permeable container contains cells including IL-2, 2X antigen-presenting feeder cells, and OKT-3 Medium.

In some embodiments, the method comprises obtaining a tumor tissue sample from a mammal during the method; culturing the tumor tissue sample in a first gas-permeable container for a duration of about 1 to 7 days (e.g., about 7 days) as the initial first expansion , the first gas-permeable container containing cell culture medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring TILs to a second gas-permeable container and expanding the number of TILs in the second gas-permeable container for about 7 to 14 days or about 7 to 9 days, such as about 7 days, about 8 days or about 9 days, about 10 days or about 11 days , the second air-permeable container contains IL-2, 2X antigen presenting feeder cells and OKT-3 cell culture medium.

In some embodiments, the method comprises obtaining a tumor tissue sample from a mammal during the method; culturing the tumor tissue sample in a first gas-permeable container for a duration of about 1 to 7 days (e.g., about 7 days) as the initial first expansion , the first gas-permeable container containing cell culture medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3; transferring TILs to a second gas-permeable container and expanding the number of TILs in the second gas-permeable container for about 7 to 11 days, eg, about 7 days, about 8 days, about 9 days, about 10 days, or about 11 days, the second gas permeable container contains cell culture medium comprising IL-2, 2X antigen presenting feeder cells, and OKT-3.

In some embodiments, TILs are expanded in gas-permeable containers. Gas-permeable containers have been used to expand TILs, using PBMCs, using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. 2005/0106717 A1, the disclosure of which is incorporated by reference way incorporated into this article. In some embodiments, TILs are expanded in gas permeable bags. In some embodiments, TILs are expanded using a cell expansion system that expands TILs in a gas-permeable bag, such as the Xuri Cell Expansion System W25 (GE Healthcare). In some embodiments, TILs are expanded using a cell expansion system that expands TILs in a gas-permeable bag, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In some embodiments, the cell expansion system includes a gas permeable cell bag having a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L and about 10 L.

In some embodiments, TILs can be expanded in G-REX flasks (available from Wilson Wolf Manufacturing). Such embodiments allow expansion of cell populations from about 5 x ¹⁰⁵ cells/cm2 to between 10 x ¹⁰⁶ and 30 x ¹⁰⁶ cells/cm2. In some embodiments, the line is not fed. In some embodiments, there is no feeding, as long as the medium in the G-REX flask resides at a height of about 10 cm. In some embodiments, this is without feeding but with the addition of one or more cytokines. In some embodiments, the cytokines can be added as a bolus without mixing the cytokines with the medium. Such vessels, devices and methods are known in the art and have been used to expand TILs, and include those described in: U.S. Patent Application Publication No. US 2014/0377739A1 , International Publication No. WO 2014/210036 A1, U.S. Patent Application Publication No. us 2013/0115617 A1, International Publication No. WO 2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228 A1, U.S. Patent No. US 8,809,050 B2 , International Publication No. WO 2011/072088 A2, U.S. Patent Application Publication No. US 2016/0208216 A1, U.S. Patent Application Publication No. US 2012/0244133 A1, International Publication No. WO 2012/129201 A1, U.S. Patent Application Publication No. US 2013/0102075 A1, U.S. Patent No. US 8,956,860 B2, International Publication No. WO 2013/173835 A1, and U.S. Patent Application Publication No. US 2015/0175966 A1, the disclosures of which are incorporated herein by reference way incorporated into this article. Such a process is also described in Jin et al., Journal of Immunotherapy, 2012, 35:283-292. D. Optional gene attenuation or gene knockout of genes in TIL

In some embodiments, the expanded TILs of the invention are further manipulated to alter protein expression in a transient manner before, during, or after the amplification step, including during closed aseptic manufacturing procedures, each as provided herein. In some embodiments, the temporarily altered protein expression is due to temporary gene editing. In some embodiments, the expanded TILs of the invention are treated with transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression in TILs. In some embodiments, TF and/or other molecules capable of temporarily altering protein expression provide altered tumor antigen expression and/or alter the number of tumor antigen-specific T cells in a population of TILs.

In certain embodiments, the methods comprise gene editing a population of TILs. In certain embodiments, the method comprises gene editing the first population of TILs, the second population of TILs and/or the third population of TILs.

In some embodiments, the invention includes gene editing via nucleotide insertion, such as via ribonucleic acid (RNA) insertion, including insertion of messenger RNA (mRNA) or small (or short) interfering RNA (siRNA), into a population of TILs, To promote the expression of one or more proteins or inhibit the expression of one or more proteins and simultaneously promote the combination of one group of proteins and inhibit another group of proteins.

In some embodiments, the expanded TILs of the invention undergo transient changes in protein expression. In some embodiments, the transient change in protein expression occurs in the subject TIL population prior to the first amplification, including, for example, obtained from, for example, FIG. 8 (especially FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) indicated in the TIL population of step A. In some embodiments, a transient change in protein expression occurs during the first amplification, including, for example, obtained from, for example, FIG. 8 (eg, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G) indicated the TIL population of Step B. In some embodiments, a transient change in protein expression occurs after the first expansion, including, for example, a population of TILs that transitions between the first and second amplifications (e.g., the second population of TILs described herein), That is the population of TILs obtained eg from step B as indicated in Figure 8 and included in step C. In some embodiments, the transient change in protein expression occurs in the subject TIL population prior to the second expansion, including, for example, the TIL population obtained from step C and expanded in step D, eg as indicated in FIG. 8 . In some embodiments, the transient change in protein expression occurs during a second expansion, including, for example, a population of TILs amplified in step D, eg, as indicated in FIG. 8 (eg, a third population of TILs). In some embodiments, the transient change in protein expression occurs after a second expansion, including, for example, in the expanded TIL population obtained in step D, eg as indicated in FIG. 8 .

In some embodiments, the method of temporarily altering protein expression in a population of TILs comprises the step of electroporation. Electroporation methods are known in the art and described, for example, in Tsong, Biophys. Magazine 1991, 60, 297-306 and US Patent Application Publication No. 2014/0227237 A1 , the respective disclosures of which are incorporated herein by reference. In some embodiments, the method of temporarily altering protein expression in a TIL population comprises the step of calcium phosphate transfection. Calcium phosphate transfection method (calcium phosphate DNA precipitation , cell surface coating and endocytosis) are known in the art and described in: Graham and van der Eb, "Virology (Virology)" 1973, 52, 456-467; Wigler et al., " Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell . Biol . 1987, 7, 2745-2752 and U.S. Patent No. 5,593,875, the respective disclosures of which are incorporated herein by reference. In some embodiments, the method of temporarily altering protein expression in a TIL population comprises the step of lipofection. Lipofectamine transfection dyeing methods, such as the use of cationic lipids N- [1-(2,3-dioleyloxy)propyl]-n ,n,n -trimethylammonium chloride (DOTMA) and dioleylphosphatidyl The method of 1:1 (w/w) liposome formulation of ethanolamine (DOPE) in filtered water is known in the art and described in: Rose et al., Biotechniques 1991, 10 , 520-525 and Felgner et al., Proceedings of the National Academy of Sciences, 1987, 84, 7413-7417 and US Patent Nos. 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070 , the respective disclosures of which are incorporated herein by reference. In some embodiments, the method of temporarily altering protein expression in a TIL population comprises a transfection step using the method described in: U.S. Patent No. 5,766,902, Nos. 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of each of which are incorporated herein by reference.

In some embodiments, temporarily altering protein expression results in an increase in Stem Memory T cells (TSCM). TSCM are early precursor cells of antigen-experienced central memory T cells. TSCMs generally exhibit long-term survival, self-renewal, and pluripotency capabilities that define stem cells, and are generally required for efficient TIL production. TSCM showed enhanced antitumor activity compared with other T cell subsets in a mouse model of recipient cell transfer. In some embodiments, temporarily altering protein expression results in a population of TILs with a composition comprising a high proportion of TSCMs. In some embodiments, temporarily altering protein expression results in an increase in percent TSCM of at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% , at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, temporarily altering protein expression results in at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in TSCM in a population of TILs. In some embodiments, the transient generation of protein expression has at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the TIL population of TSCM. In some embodiments, temporarily altering protein expression produces a protein with at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the TSCM therapeutic TIL population.

In some embodiments, temporarily altering protein expression causes the antigen to undergo T cell rejuvenation. In some embodiments, rejuvenation includes, for example, increased proliferation, increased T cell activation, and/or increased antigen recognition.

In some embodiments, temporarily altering protein expression alters the expression of a subset of T cells to preserve tumor-derived TCR repertoire. In some embodiments, temporarily altering protein expression does not alter tumor-derived TCR repertoire. In some embodiments, temporarily altering protein expression maintains a tumor-derived TCR repertoire.

In some embodiments, temporarily altering a protein results in altered expression of a particular gene. In some embodiments, temporarily altering protein expression targets genes including, but not limited to, the following: CD39, CD69, PD-1 (also known as PDCD1 or CC279), TGFBR2, CCR4/5, CBLB (CBL-B ), CISH, chimeric co-stimulatory receptor (CCR), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, CTLA-4, TIM3, LAG3, TIGIT , TET2, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2(MCP-1), CCL3(MIP-1α), CCL4(MIP1-β), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17 , CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection-associated high mobility group (high mobility group; HMG) box (TOX), ankyrin repeat domain 11 (ANKRD11), BCL6 co-repressor (BCOR) and/or cAMP protein kinase A (PKA). In some embodiments, temporarily altering protein expression targets a gene selected from the group consisting of: PD-1, TGFBR2, CCR4/5, CTLA-4, CBLB (CBL-B), CISH, chimeric co-stimulatory receptor CCR, IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TET2, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection-related high Mobility group (HMG) box (TOX), ankyrin repeat domain 11 (ANKRD11), BCL6 co-repressor (BCOR) and/or cAMP protein kinase A (PKA). In some embodiments, temporarily altering protein expression targets CD39. In some embodiments, temporarily altering protein expression targets CD69. In some embodiments, temporarily altering protein expression targets PD-1. In some embodiments, the temporarily altered protein expression targets TGFBR2. In some embodiments, temporarily altering protein expression targets CCR4/5. In some embodiments, temporarily altering protein expression targets CTLA-4. In some embodiments, temporarily altering protein expression targets CBLB. In some embodiments, temporarily altering protein expression targets CISH. In some embodiments, temporarily altering protein expression targets a CCR (chimeric co-stimulatory receptor). In some embodiments, temporarily altering protein expression targets IL-2. In some embodiments, temporarily altering protein expression targets IL-12. In some embodiments, temporarily altering protein expression targets IL-15. In some embodiments, temporarily altering protein expression targets IL-21. In some embodiments, temporarily altering protein expression targets NOTCH 1/2 ICD. In some embodiments, temporarily altering protein expression targets TIM3. In some embodiments, temporarily altering protein expression targets LAG3. In some embodiments, temporarily altering protein expression targets TIGIT. In some embodiments, temporarily altering protein expression targets TET2. In some embodiments, temporarily altering protein expression targets TGFβ. In some embodiments, temporarily altering protein expression targets CCR1. In some embodiments, temporarily altering protein expression targets CCR2. In some embodiments, temporarily altering protein expression targets CCR4. In some embodiments, temporarily altering protein expression targets CCR5. In some embodiments, temporarily altering protein expression targets CXCR1. In some embodiments, temporarily altering protein expression targets CXCR2. In some embodiments, temporarily altering protein expression targets CSCR3. In some embodiments, the temporarily altered protein expression targets CCL2 (MCP-1). In some embodiments, the temporarily altered protein expression targets CCL3 (MIP-1α). In some embodiments, temporarily altering protein expression targets CCL4 (MIP1-beta). In some embodiments, the transiently altered protein expression targets CCL5 (RANTES). In some embodiments, temporarily altering protein expression targets CXCL1. In some embodiments, temporarily altering protein expression targets CXCL8. In some embodiments, temporarily altering protein expression targets CCL22. In some embodiments, temporarily altering protein expression targets CCL17. In some embodiments, temporarily altering protein expression targets VHL. In some embodiments, temporarily altering protein expression targets CD44. In some embodiments, temporarily altering protein expression targets PIK3CD. In some embodiments, temporarily altering protein expression targets SOCS1. In some embodiments, temporarily altering protein expression targets the high mobility group (HMG) cassette (TOX) associated with thymocyte selection. In some embodiments, the temporarily altered protein expression targets ankyrin repeat domain 11 (ANKRD11). In some embodiments, temporarily altering protein expression targets the BCL6 corepressor (BCOR). In some embodiments, the temporarily altered protein expression targets cAMP protein kinase A (PKA).

In some embodiments, temporarily altering protein expression results in increased and/or overexpressed chemokine receptors. In some embodiments, chemokine receptors overexpressed by transient protein expression include receptors with ligands including, but not limited to, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8, CCL22 and/or CCL17.

In some embodiments, temporarily altering protein expression results in decreased expression of CD39, CD69, PD-1, CTLA-4, CBLB, CISH, TIM-3, LAG-3, TIGIT, TET2, TGFβR2 and/or TGFβ and/or or decrease (including causing eg TGFβ pathway block). In some embodiments, temporarily altering protein expression results in decreased expression and/or reduction of PD-1. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CBLB (CBL-B). In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CISH. In some embodiments, temporarily altering protein expression results in decreased expression and/or reduction of TIM-3. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of LAG-3. In some embodiments, temporarily altering protein expression results in decreased and/or diminished expression of TIGIT. In some embodiments, temporarily altering protein expression results in decreased expression and/or reduction of TET2. In some embodiments, temporarily altering protein expression results in decreased expression and/or reduction of TGFβR2. In some embodiments, temporarily altering protein expression results in decreased expression and/or reduction of TGF[beta].

In some embodiments, temporarily altering protein expression results in increased and/or overexpressed chemokine receptors, eg, to improve TIL trafficking or motility to the tumor site. In some embodiments, temporarily altering protein expression results in increased and/or overexpressed chimeric co-stimulatory receptors (CCRs). In some embodiments, temporarily altering protein expression results in increased and/or overexpressed chemokine receptors selected from the group consisting of: CCR1 , CCR2, CCR4, CCR5, CXCR1 , CXCR2 and/or CSCR3.

In some embodiments, temporarily altering protein expression results in increased interleukin and/or overexpression. In some embodiments, temporarily altering protein expression results in increased and/or overexpression of an interleukin selected from the group consisting of: IL-2, IL-12, IL-15, and/or IL-21.

In some embodiments, temporarily altering protein expression results in increased and/or overexpression of NOTCH 1/2 ICD. In some embodiments, temporarily altering protein expression results in increased and/or overexpressed VHL. In some embodiments, temporarily altering protein expression results in increased and/or overexpressed CD44. In some embodiments, temporarily altering protein expression results in increased and/or overexpression of PIK3CD. In some embodiments, temporarily altering protein expression results in increased and/or overexpressed SOCS1.

In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of cAMP protein kinase A (PKA).

In some embodiments, temporarily altering protein expression results in decreased expression and/or decreased expression of a molecule selected from the group consisting of: CD39, CD69, PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH , TGFβR2, PKA, CBLB, BAFF (BR3) and combinations thereof. In some embodiments, temporarily altering protein expression results in decreased expression and/or decreased expression of two molecules selected from the group consisting of: CD39, CD69, PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2 , CISH, TGFβR2, PKA, CBLB, BAFF (BR3) and combinations thereof. In some embodiments, temporarily altering protein expression results in decreased expression and/or decreased expression of PD-1 and a molecule selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PKA , CBLB, BAFF (BR3) and combinations thereof. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of PD-1, CTLA-4, LAG-3, CISH, CBLB, TIM3, TIGIT, TET2, and combinations thereof. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of PD-1 and one of: CTLA-4, LAG3, CISH, CBLB, TIM3, TIGIT, TET2, and combinations thereof. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of PD-1 and CTLA-4. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of PD-1 and LAG3. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of PD-1 and CISH. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of PD-1 and CBLB. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CD39 and CD69. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of PD-1 and TIM3. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of PD-1 and TIGIT. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of PD-1 and TET2. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CTLA-4 and LAG3. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CTLA-4 and CISH. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CTLA-4 and CBLB. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CTLA-4 and TIM3. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CTLA-4 and TIGIT. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CTLA-4 and TET2. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of LAG3 and CISH. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of LAG3 and CBLB. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of LAG3 and TIM3. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of LAG3 and TIGIT. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of LAG3 and TET2. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CISH and CBLB. In some embodiments, temporarily altering protein expression results in decreased and/or reduced expression of CISH and TIM3. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CISH and TIGIT. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CISH and TET2. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CBLB and TIM3. In some embodiments, temporarily altering protein expression results in decreased and/or reduced expression of CBLB and TIGIT. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of CBLB and TET2. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of TIM3 and PD-1. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of TIM3 and LAG3. In some embodiments, temporarily altering protein expression results in reduced and/or decreased expression of TIM3 and CISH. In some embodiments, temporarily altering protein expression results in reduced and/or decreased expression of TIM3 and CBLB. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of TIM3 and TIGIT. In some embodiments, temporarily altering protein expression results in reduced and/or reduced expression of TIM3 and TET2.

In some embodiments, an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof is inserted by gamma retroviral or lentiviral methods into the first TIL population, the second TIL population, or in the collected TIL population (eg increased expression of adhesion molecules).

In some embodiments, temporarily altering protein expression causes a protein selected from the group consisting of CD39, CD69, PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. The expression of the molecules of the group is decreased and/or decreased, and the expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof is increased and/or enhanced. In some embodiments, temporarily altering protein expression results in decreased expression of a molecule selected from the group consisting of CD39, CD69, PD-1, CTLA-4, LAG3, TIM3, CISH, CBLB, TIGIT, TET2, and combinations thereof and and/or decreased, and increased and/or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

In some embodiments, the performance is reduced by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55% %, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in performance is at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in performance is at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in performance is at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, performance is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, performance is reduced by at least about 80%. In some embodiments, performance is reduced by at least about 85%. In some embodiments, performance is reduced by at least about 90%. In some embodiments, performance is reduced by at least about 95%. In some embodiments, performance is reduced by at least about 99%.

In some embodiments, the performance is increased by about 5% , about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55% %, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the increase in performance is at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the increase in performance is at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the increase in performance is at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, performance is increased by at least about 85%, about 90%, or about 95%. In some embodiments, performance is increased by at least about 80%. In some embodiments, performance is increased by at least about 85%. In some embodiments, performance is increased by at least about 90%. In some embodiments, performance is increased by at least about 95%. In some embodiments, performance is increased by at least about 99%.

In some embodiments, transiently altering protein expression is induced by treating TIL with transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression in TIL. In some embodiments, microfluidic platforms without SQZ vectors are used for intracellular delivery of transcription factors (TFs) and/or other molecules capable of temporarily altering protein expression. Such methods of demonstrating the ability to deliver proteins, including transcription factors, to a variety of primary human cells, including T cells, have been described in: US Patent Application Publication No. 2019/0093073 A1, US 2018/0201889 No. A1 and US 2019/0017072 No. A1, the respective disclosures of which are incorporated herein by reference. Such methods can be used in the present invention to expose TIL populations to transcription factors (TFs) and/or other molecules capable of inducing transient protein expression, wherein the TFs and/or other molecules capable of inducing transient protein expression provide Increased expression of tumor antigens in the TIL population and/or increased numbers of tumor antigen-specific T cells results in reprogramming of the TIL population and increased therapeutic efficacy of the reprogrammed TIL population compared to non-reprogrammed TIL populations. In some embodiments, reprogramming results in an increase in effector T cell and/or central memory T cell subsets relative to a starting or previous TIL population (ie, prior to reprogramming), as described herein.

In some embodiments, transcription factors (TFs) include, but are not limited to, TCF-1, NOTCH 1/2 ICD, and/or MYB. In some embodiments, the transcription factor (TF) is TCF-1. In some embodiments, the transcription factor (TF) is NOTCH 1/2 ICD. In some embodiments, the transcription factor (TF) is MYB. In some embodiments, transcription factors (TFs) are administered with induced potential-rich stem cell cultures (iPSCs), such as commercially available KNOCKOUT serum replacement (Gibco/Thermo Fisher), to induce additional TIL reprogramming. In some embodiments, transcription factors (TFs) are administered with the iPSC mixture to induce additional TIL reprogramming. In some embodiments, transcription factors (TFs) are not administered with the iPSC mixture. In some embodiments, reprogramming results in an increased percentage of TSCM. In some embodiments, the reprogramming increases the percentage of TSCM by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, About 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% TSCM.

In some embodiments, methods of temporarily altering protein expression as described above may be combined with methods of genetically modifying TIL populations, including the step of stably incorporating genes to produce one or more proteins. In certain embodiments, the methods comprise the step of genetically modifying a population of TILs. In certain embodiments, the method comprises genetically modifying the first population of TILs, the second population of TILs and/or the third population of TILs. In some embodiments, the method of genetically modifying a population of TILs includes the step of retroviral transduction. In some embodiments, the method of genetically modifying a population of TILs includes the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in: Levine et al., Proceedings of the National Academy of Sciences USA 2006, 103, 17372-77; Zufferey et al., Nature Biotechnology 1997 , 15, 871-75; Dull et al., Journal of Virology 1998, 72, 8463-71 and US Patent No. 6,627,442, the respective disclosures of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes the step of gamma-retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, Current Protocols in Molecular Biology (Cur. Case) 1996, 9.9.1-9.9.16, the disclosure of which Incorporated herein by reference. In some embodiments, the method for genetically modifying a TIL population includes the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art, and Included are those in which the translocase is provided as a DNA expression vector or as an expressible RNA or protein such that long-term expression of the translocase does not occur in transgenic cells, such as provided as mRNA (e.g., comprising a cap and a polyA tail mRNA) translocases. Including salmon-like Tel-like translocases (SB or Sleeping Beauty translocases), such as SB10, SB11 and SB100x; Gene transfer systems are described, for example, in Hackett et al., Molecular Therapy 2010, 18, 674-83 and US Patent No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

In some embodiments, the temporary alteration of protein expression in TIL is induced by small interfering RNA (small interfering RNA; siRNA), sometimes referred to as short interfering RNA or silencing RNA, which is a double-stranded RNA molecule, Typically 19-25 base pairs in length. siRNA is used in RNA interference (RNA interference; RNAi), wherein siRNA interferes with the expression of a specific gene having a complementary nucleotide sequence.

In some embodiments, temporarily altering the protein manifests as a decrease in expression. In some embodiments, the transiently altered protein expression in TILs is induced by self-delivering RNA interference (sdRNA) that is sdRNA with a high percentage of 2' -OH substitutions (typically fluorine or -OCH3) Chemically synthesized asymmetric siRNA duplex comprising a 20 nucleotide antisense (guide) strand and 13 to 15 nucleotides conjugated to cholesterol at its 3' end using a tetraethylethylene glycol (TEG) linker Base sense (passenger) strands. Small interfering RNA (siRNA), sometimes called short interfering RNA or silencing RNA, is a double-stranded RNA molecule, typically 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), in which siRNA interferes with the expression of a specific gene with a complementary nucleotide sequence. sdRNAs are covalently and hydrophobically modified RNAi compounds that do not require a delivery vehicle to enter cells. sdRNA is generally an asymmetric chemically modified nucleic acid molecule with a very small double-stranded region. sdRNA molecules typically contain single- and double-stranded regions, and can contain various chemical modifications within the single- and double-stranded regions of the molecule. Additionally, as described herein, sdRNA molecules can be linked to hydrophobic binders, such as conventional and higher sterol-type molecules. sdRNA and related methods of making such sdRNA have also been extensively described in, for example, U.S. Patent Application Publication Nos. US 2016/0304873 A1 , US 2019/0211337 A1 , US 2009/0131360 A1 , and US 2019 /0048341 A1, and US Patent Nos. 10,633,654 and 10,913,948 B2, the respective disclosures of which are incorporated herein by reference. To optimize sdRNA structure, chemical properties, targeting location, sequence preference and the like, an algorithm has been developed and used for sdRNA potency prediction. Based on these analyses, a functional sdRNA sequence is generally defined as exhibiting greater than 70% reduction at a concentration of 1 µM, with a probability greater than 40%.

Double-stranded DNA (dsRNA) can generally be used to define any molecule comprising a pair of complementary RNA strands, typically a sense (passenger) and antisense (guide) strand, and can include a single-stranded overhanging arm region. In contrast to siRNA, the term dsRNA generally refers to precursor molecules comprising sequences of siRNA molecules released from larger dsRNA molecules by the action of cleavage enzyme systems, including Dicer.

In some embodiments, the method comprises temporarily altering protein expression in a population of TILs, including TILs modified to express a CCR, including the use of self-delivering RNA interference (sdRNA), e.g., having a higher percentage of 2'- OH-substituted (typically fluorine or OCH ₃ ) chemically synthesized asymmetric siRNA duplex comprising a 20 nucleotide antisense (guide) strand with a tetraethylethylene glycol (TEG) linker in its A 13 to 15 base sense (passenger) strand bound to cholesterol at the 3' end. Methods using siRNA and sdRNA have been described in: Khvorova and Watts, Nat. Biotechnol. 2017, 35 , 238-248; Byrne et al., Journal of Ophthalmic Pharmacology and Therapeutics ( J . Ocul. Pharmacol. Ther. ) 2013, 29, 855-864; and Ligtenberg et al., Molecular Therapy 2018 , 26, 1482-93 , the disclosures of which are incorporated herein by reference. In one embodiment, delivery of siRNA is accomplished using electroporation or cell membrane disruption such as extrusion or SQZ methods. In some embodiments, delivery of sdRNA to TIL populations does not need to be accomplished using electroporation, SQZ, or other methods, and is actually accomplished using a 1 to 3 day period to expose TIL populations to sdRNA at a concentration of 1 µM/10,000 TILs in culture medium . In certain embodiments, the method comprises delivering siRNA or sdRNA to a population of TILs comprising exposing the population of TILs to the sdRNA at a concentration of 1 μΜ/10,000 TILs in culture medium for a period of between 1 and 3 days. In some embodiments, delivery of sdRNA to TIL populations is accomplished using a period of 1 to 3 days in which the TIL populations are exposed to sdRNA at a concentration of 10 μM/10,000 TILs in culture medium. In some embodiments, delivery of sdRNA to TIL populations is accomplished using a period of 1 to 3 days in which the TIL populations are exposed to sdRNA at a concentration of 50 μM/10,000 TILs in culture medium. In some embodiments, delivery of sdRNA to a population of TILs is accomplished using a period of 1 to 3 days in which the population of TILs is exposed to the sdRNA at a concentration in culture medium between 0.1 µM/10,000 TILs and 50 µM/10,000 TILs. In some embodiments, delivery of the sdRNA to the TIL population is accomplished using a 1 to 3 day period in which the TIL population is exposed to the sdRNA at a concentration in culture medium between 0.1 µM/10,000 TIL and 50 µM/10,000 TIL, Wherein the exposure to sdRNA is performed two, three, four or five times by adding fresh sdRNA to the culture medium. Other suitable processes are described, for example, in U.S. Patent Application Publication Nos. US 2011/0039914 A1 , US 2013/0131141 A1 , and US 2013/0131142 A1 , and U.S. Patent No. 9,080,171 , the disclosures of which are incorporated by reference way incorporated into this article.

In some embodiments, the siRNA or sdRNA is inserted into the TIL population during manufacturing. In certain embodiments, the sdRNA encoding interferes with NOTCH 1/2 ICD, PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFβ, TGFBR2, cAMP protein kinase A (PKA), BAFF BR3, CISH and/or CBLB RNA. In some embodiments, reduction in expression is determined based on the percentage of gene silencing, eg, as assessed by flow cytometry and/or qPCR. In some embodiments, the performance is reduced by about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55% %, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in performance is at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in performance is at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in performance is at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, performance is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, performance is reduced by at least about 80%. In some embodiments, performance is reduced by at least about 85%. In some embodiments, performance is reduced by at least about 90%. In some embodiments, performance is reduced by at least about 95%. In some embodiments, performance is reduced by at least about 99%.

Self-delivery RNAi technology based on chemical modification of siRNA can be used with the methods of the present invention to successfully deliver sdRNA to TILs as described herein. Combinations of backbone modifications with asymmetric siRNA structures and hydrophobic ligands (see, for example, Ligtenberg et al., Molecular Therapy 2018, 26 , 1482-93 and US Patent Application Publication No. 2016/0304873 A1, the disclosures of which are incorporated herein by reference) allows sdRNAs to penetrate cultured mammalian cells by simply adding them to the culture medium, taking advantage of the nuclease stability of sdRNAs, without the need for additional formulations and methods. This stability allows a constant level of RNAi-mediated reduction in target gene activity to be supported simply by maintaining the active concentration of sdRNA in the culture medium. While not being bound by theory, backbone stabilization of the sdRNA results in a prolonged gene expression reduction effect that can last for months in non-dividing cells.

In some embodiments, the transfection efficiency of TIL exceeds 95%, and there is a decrease in target expression caused by each specific siRNA or sdRNA. In certain embodiments, an siRNA or sdRNA containing several unmodified ribose residues is replaced with a fully modified sequence to increase the potency and/or persistence of the RNAi effect. In some embodiments, the performance-reducing effect is maintained for 12 hours, 24 hours, 36 hours, 48 hours, 5 days, 6 days, 7 days, or 8 days or more. In some embodiments, the performance-reducing effect is reduced 10 days or more after siRNA or sdRNA treatment of the TIL. In some embodiments, the target performance is maintained with a performance reduction of greater than 70%. In some embodiments, target performance in the TIL is maintained with a performance reduction of greater than 70%. In some embodiments, reduced expression in the PD-1/PD-L1 pathway allows TILs to exhibit more potent in vivo effects, in some embodiments by avoiding the inhibitory effects of the PD-1/PD-L1 pathway. In some embodiments, reduction of PD-1 expression by siRNA or sdRNA results in increased TIL proliferation.

In some embodiments, the sdRNA sequences used in the invention exhibit a 70% reduction in the expression of the gene of interest. In some embodiments, the sdRNA sequences used in the invention exhibit a 75% reduction in the expression of the gene of interest. In some embodiments, the sdRNA sequences used in the invention exhibit an 80% reduction in the expression of the gene of interest. In some embodiments, the sdRNA sequences used in the invention exhibit an 85% reduction in the expression of the gene of interest. In some embodiments, the sdRNA sequences used in the invention exhibit a 90% reduction in the expression of the gene of interest. In some embodiments, the sdRNA sequences used in the invention exhibit a 95% reduction in the expression of the gene of interest. In some embodiments, the sdRNA sequences used in the invention exhibit a 99% reduction in the expression of the gene of interest. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of a gene of interest when delivered at a concentration of about 0.25 µM to about 4 µM. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the gene of interest when delivered at a concentration of about 0.25 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the gene of interest when delivered at a concentration of about 0.5 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the gene of interest when delivered at a concentration of about 0.75 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in target gene expression when delivered at a concentration of about 1.0 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the gene of interest when delivered at a concentration of about 1.25 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the gene of interest when delivered at a concentration of about 1.5 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in target gene expression when delivered at a concentration of about 1.75 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in target gene expression when delivered at a concentration of about 2.0 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the gene of interest when delivered at a concentration of about 2.25 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the gene of interest when delivered at a concentration of about 2.5 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in target gene expression when delivered at a concentration of about 2.75 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the gene of interest when delivered at a concentration of about 3.0 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the gene of interest when delivered at a concentration of about 3.25 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in target gene expression when delivered at a concentration of about 3.5 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the gene of interest when delivered at a concentration of about 3.75 μΜ. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in target gene expression when delivered at a concentration of about 4.0 μΜ.

In some embodiments, siRNA or sdRNA oligonucleotide agents comprise one or more modifications that increase the stability and/or effectiveness of the therapeutic agent and enable efficient delivery of the oligonucleotide to the cell or tissue to be treated. Such modifications may include 2'-O-methyl modifications, 2'-O-fluoro modifications, phosphorodithioate modifications, 2'F modified nucleotides, 2'-O-methyl modified nucleotides And/or 2' deoxynucleotides. In some embodiments, oligonucleotides are modified to include one or more hydrophobic modifications including, for example, sterol, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and / or phenyl. In some embodiments, the chemically modified nucleotides are a combination of phosphorothioate, 2'-O-methyl, 2'deoxy, hydrophobic modification, and phosphorothioate. In some embodiments, sugars may be modified and modified sugars may include, but are not limited to, D-ribose, 2'-O-alkyl (including 2'-O-methyl and 2'-O-ethyl), That is, 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halo (including 2'-fluoro), T-methoxyethoxy, 2'-ene Propoxy (-OCH ₂ CH=CH ₂ ), 2'-propargyl, 2'-propyl, ethynyl, vinyl, propenyl, cyano and the like. In some embodiments, the sugar moiety may be a hexose sugar and incorporated into an oligonucleotide, such as Augustyns et al., Nucl. Acids. Res., 1992, 18 , 4711, the disclosure of which is cited by reference way incorporated into this article.

In some embodiments, double-stranded siRNA or sdRNA oligonucleotides of the invention are double-stranded throughout their length, ie, have no overhanging single-stranded sequences at either end of the molecule, ie, are blunt-ended. In some embodiments, individual nucleic acid molecules can be of different lengths. In other words, the double-stranded siRNA or sdRNA oligonucleotides of the invention are not double-stranded throughout their length. For example, when two separate nucleic acid molecules are used, one of the molecules, eg, the first molecule comprising the antisense sequence, may be longer than the second molecule to which it hybridizes (leaving a portion of the molecule as a single strand). In some embodiments, when using single nucleic acid molecules, a portion of the molecule at either end can remain single stranded.

In some embodiments, double-stranded siRNA or sdRNA oligonucleotides of the invention contain mismatches and/or loops or bulges, but are double-stranded over at least about 70% of the length of the oligonucleotide. In some embodiments, double-stranded oligonucleotides of the invention are double-stranded for at least about 80% of the length of the oligonucleotide. In other embodiments, double-stranded siRNA or sdRNA oligonucleotides of the invention are double-stranded at least about 90%-95% of the length of the oligonucleotide. In some embodiments, double-stranded siRNA or sdRNA oligonucleotides of the invention are double-stranded at least about 96%-98% of the length of the oligonucleotide. In some embodiments, the double-stranded oligonucleotides of the invention contain at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 errors. match.

In some embodiments, siRNA or sdRNA oligonucleotides can be substantially protected from nucleases, such as by modifying the 3' or 5' linkages (eg, U.S. Patent No. 5,849,902, the disclosure of which is incorporated by reference Incorporated herein. For example, oligonucleotides can be resistant by incorporating a "blocking group". As used herein, the term "blocking group" refers to a protective group that can be used for synthesis or Substituents (for example other than OH groups) (for example FITC, propyl (CH ₂ -CH ₂ -CH ₃ ), diol (-0-CH ₂ - CH ₂ -O-) phosphate (PO ₃ ^2" ), hydrogen phosphonate or phosphoramidate). "Blocking group" may also include "terminal blocking group" or "exonuclease blocking group""Blockinggroup" which protects the 5' and 3' ends of an oligonucleotide, which includes modified nucleotide and non-nucleotide exonuclease resistant structures.

In some embodiments, at least a portion of contiguous polynucleotides within the siRNA or sdRNA are linked by substituent linkages, such as phosphorothioate linkages.

In some embodiments, the chemical modification results in at least 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% cellular uptake of the siRNA or sdRNA , 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100 %, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500% enhancement. In some embodiments, at least one of the C or U residues includes a hydrophobic modification. In some embodiments, the plurality of C and U contain hydrophobic modifications. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of C and U can contain hydrophobic modification. In some embodiments, all C and U contain hydrophobic modifications.

In some embodiments, siRNA or sdRNA molecules exhibit enhanced release from endosomes via incorporation of protonatable amines. In some embodiments, protonatable amines are incorporated into the sense strand (in the portion of the molecule that is discarded after RISC loading). In some embodiments, siRNA or sdRNA compounds of the invention comprise asymmetric compounds comprising a duplex region (required for efficient RISC entry, 10-15 bases long) and 4-12 nucleotides long Single-stranded region; double helix with 13 nucleotides. In some embodiments, single-stranded regions of 6 nucleotides are employed. In some embodiments, the single-stranded region of the siRNA or sdRNA comprises 2-12 phosphorothioate internucleotide linkages (referred to as phosphorothioate modifications). In some embodiments, 6-8 phosphorothioate internucleotide linkages are employed. In some embodiments, siRNA or sdRNA compounds of the invention also include unique chemical modification patterns that provide stability and are compatible with RISC entry. For example, the guide strand can also be modified by any chemical modification that demonstrates stability without interfering with RISC entry. In some embodiments, the chemical modification pattern in the guide strand includes C and U nucleotides that are mostly 2'F modified and phosphorylated at the 5' end.

In some embodiments, at least 30% of the nucleotides in the siRNA or sdRNA are modified. In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% , 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotides are modified. In some embodiments, 100% of the nucleotides in the siRNA or sdRNA are modified.

In some embodiments, siRNA or sdRNA molecules have very few double-stranded regions. In some embodiments, the double-stranded region of the molecule ranges from 8-15 nucleotides in length. In some embodiments, the double-stranded region of the molecule is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides in length. In some embodiments, the double-stranded region is 13 nucleotides long. There can be 100% complementarity between the lead and passenger shares, or there can be one or more mismatches between the lead and passenger shares. In some embodiments, the molecule is blunt-ended or has an overhanging arm of one nucleotide at one end of the double-stranded molecule. The single-stranded region of the molecule is, in some embodiments, between 4-12 nucleotides in length. In some embodiments, single-stranded regions may be 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides in length. In some embodiments, single-stranded regions can also be less than 4 nucleotides or greater than 12 nucleotides long. In certain embodiments, the single-stranded region is 6 or 7 nucleotides in length.

In some embodiments, the siRNA or sdRNA molecule has increased stability. In some cases, the half-life of the chemically modified siRNA or sdRNA molecule in culture medium is longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more than 24 hours, including any intermediate values. In some embodiments, the half-life of the siRNA or sd-RNA in culture medium is greater than 12 hours.

In some embodiments, siRNA or sdRNA are optimized to increase potency and/or reduce toxicity. In some embodiments, the nucleotide length of the guide strand and/or passenger strand and/or the number of phosphorothioate modifications in the guide strand and/or passenger strand can affect the performance of the RNA molecule in some aspects, and using The substitution of 2'-O-methyl (2'OMe) modification for 2'-fluoro (2'F) modification can affect the toxicity of the molecule in some aspects. In some embodiments, reducing the 2'F content of a molecule is expected to reduce the toxicity of the molecule. In some embodiments, the number of phosphorothioate modifications in an RNA molecule can affect the efficiency of uptake of the molecule into the cell, eg, passive uptake of the molecule into the cell. In some embodiments, the siRNA or sdRNA does not have a 2'F modification, but is characterized by equal efficacy in terms of cellular uptake and tissue penetration.

In some embodiments, the guide strand is about 18-19 nucleotides in length and has about 2-14 phosphate modifications. For example, a leader strand can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 phosphate-modified nucleotides. The guide strand may contain one or more modifications that confer increased stability without interfering with RISC entry. Phosphate-modified nucleotides, such as phosphorothioate-modified nucleotides, can be at the 3' end, the 5' end, or throughout the leader strand. In some embodiments, the 3' 10 nucleotides of the leading strand contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioate modified nucleotides. The guide strand may also contain 2'F and/or 2'OMe modifications, which may be located throughout the molecule. In some embodiments, the nucleotide in position one of the lead strand (the nucleotide in the most 5' position of the lead strand) is 2'OMe modified and/or phosphorylated. C and U nucleotides within the lead strand can be 2'F modified. For example, the C and U nucleotides in positions 2-10 of the 19 nucleotide lead strand (or the corresponding positions in lead strands of different lengths) can be 2'F modified. The C and U nucleotides in the lead strand can also be modified with 2'OMe. For example, the C and U nucleotides in positions 11-18 of the 19 nucleotide lead strand (or the corresponding positions in lead strands of different lengths) can be 2'OMe modified. In some embodiments, the nucleotides at the most 3' end of the leading strand are unmodified. In certain embodiments, most of the C and U within the leading strand are 2'F modified, and the 5' end of the leading strand is phosphorylated. In other embodiments, the C or U in position 1 and positions 11-18 are 2'OMe modified and the 5' end of the leading strand is phosphorylated. In other embodiments, the C or U in position 1 and positions 11-18 are modified with 2'OMe, the 5' end of the leading strand is phosphorylated, and the C or U in positions 2-10 are modified with 2'F.

Self-deliverable RNAi technology provides a means to directly transfect cells with an RNAi agent (whether siRNA, sdRNA or other RNAi agent) without the need for additional formulations or techniques. The ability to transfect difficult-to-transfect cell lines, high in vivo activity, and ease of use are the characteristics of these compositions and methods, which present significant functional advantages over traditional siRNA-based techniques, and are therefore relevant in reducing TIL of the present invention. In some embodiments of the method for expressing a target gene, the sdRNA method is used. The sdRNA approach allows the direct delivery of chemically synthesized compounds to a wide range of primary cells and tissues in vitro and in vivo. The sdRNAs described herein in some embodiments of the invention are commercially available from Advirna LLC, Worcester, MA, USA.

siRNA and sdRNA can be formed in the form of hydrophobically modified siRNA-antisense oligonucleotide hybrid structures and are disclosed, for example, in Byrne et al., J. Ocular Pharmacol. Therapeut. , 2013, 29, 855-864, the disclosures of which are incorporated herein by reference.

In some embodiments, siRNA or sdRNA oligonucleotides can be delivered to the TILs described herein using sterile electroporation. In certain embodiments, the method comprises sterile electroporating a population of TILs to deliver siRNA or sdRNA oligonucleotides.

In some embodiments, oligonucleotides can be delivered to cells in combination with transmembrane delivery systems. In some embodiments, the transmembrane delivery system comprises lipids, viral vectors and the like. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent that does not require any delivery agent. In certain embodiments, the methods comprise using a transmembrane delivery system to deliver siRNA or sdRNA oligonucleotides to a population of TILs.

Oligonucleotides and oligonucleotide compositions are contacted (eg, contacted, also referred to herein as administered or delivered to) the TILs described herein and taken up, including passive uptake by the TIL. The sdRNA can be during the first amplification (e.g. step B), after the first amplification (e.g. during step C), before or during the second amplification (e.g. before or during step D), in step D Added after and before collection in Step E, during or after collection in Step F, before or during final dispensing and/or transfer to an infusion bag in Step F, and before any optional cryopreservation steps in Step F into TIL as described herein. Alternatively, the sdRNA can be added in Step F after thawing from any cryopreservation steps. In some embodiments, one or more genes may be targeted to genes as described herein, including CD39, CD69, PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, TET2, and CBLB. sdRNA is added to cell culture medium containing TIL and other reagents at a concentration selected from the group consisting of: 100 nM to 20 mM, 200 nM to 10 mM, 500 nM to 1 mM, 1 µM to 100 µM, and 1 µM to 100 µM. In some embodiments, one or more genes as described herein (including CD39, CD69, PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, TET2, and CBLB) can be targeted The sdRNA was added to the cell culture medium containing TIL and other reagents in an amount selected from the group consisting of: 0.1 μM sdRNA/10,000 TIL/100 μL medium, 0.5 μM sdRNA/10,000 TIL/100 μL medium, 0.75 μM sdRNA /10,000 TIL/100 μL medium, 1 μM sdRNA/10,000 TIL/100 μL medium, 1.25 μM sdRNA/10,000 TIL/100 μL medium, 1.5 μM sdRNA/10,000 TIL/100 μL medium, 2 μM sdRNA/10,000 TIL/100 μL medium, 5 μM sdRNA/10,000 TIL/100 μL medium, or 10 μM sdRNA/10,000 TIL/100 μL medium. In some embodiments, one or more of the genes as described herein (including CD39, CD69, PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, TET2CBLB) can be targeted sdRNA was added to TIL cultures twice a day, once a day, every two days, every three days, every four days, every five days, every six days, or every seven days during the pre-REP or REP phase .

Oligonucleotide compositions of the invention, including sdRNA, can be contacted with TILs as described herein during the amplification procedure, eg, by dissolving high concentrations of sdRNA in cell culture medium and allowing passive uptake for a sufficient time. In certain embodiments, the methods of the invention comprise contacting a population of TILs with an oligonucleotide composition as described herein. In certain embodiments, the method comprises dissolving the oligonucleotide, eg, sdRNA, in cell culture medium, and contacting the cell culture medium with a population of TILs. TILs can be the first population, the second population and/or the third population as described herein.

In some embodiments, delivery of oligonucleotides into cells can be enhanced by suitable art-recognized methods, including calcium phosphate, DMSO, glycerol or polydextrose, electroporation, or by transfection, e.g., using cationic , anionic or neutral lipid composition or liposomes, transfected using methods known in the art, such as those described in: U.S. Patent Nos. 4,897,355; 5,459,127; 5,631,237; 5,976,567; 10,087,464; and 10,155,945; and Bergan et al., Nucleic Acids Res. 1993, 21, 3567, the disclosures of each of which are incorporated herein by reference.

In some embodiments, more than one siRNA or sdRNA is used to reduce target gene expression. In some embodiments, one or more of CD39, CD69, PD-1, TIM-3, CBLB, LAG3, CTLA-4, TIGIT, TET2 and/or CISH targeting siRNA or sdRNA are used together. In some embodiments, PD-1 siRNA or sdRNA is used with one or more of TIM-3, CBLB, LAG3, CTLA-4, TIGIT, TET2, and/or CISH to reduce the expression of more than one gene target . In some embodiments, LAG3 siRNA or sdRNA is used in combination with CISH targeting the siRNA or sdRNA to reduce gene expression of both targets. In some embodiments, the siRNA or sdRNA targeting one or more of CD39, CD69, PD-1, TIM-3, CBLB, LAG3, CTLA-4, TIGIT, TET2 and/or CISH herein can be purchased from Advirna LLC of Worcester, MA, USA or many other suppliers.

In some embodiments, the siRNA or sdRNA targets a gene selected from the group consisting of: CD39, CD69, PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PKA, CBLB, BAFF (BR3) and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from the group consisting of: CD39, CD69, PD-1, LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PKA, CBLB, BAFF (BR3) and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1 and the other siRNA or sdRNA targets a gene selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PKA , CBLB, BAFF (BR3) and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from the group consisting of CD39, CD69, PD-1, LAG-3, CISH, CBLB, TIM3, CTLA-4, TIGIT, TET2, and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from PD-1 and one of: LAG3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets CBLB. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets TIM3. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets CBLB. In some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets TIM3. In some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets LAG3 and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets CISH and one siRNA or sdRNA targets CBLB. In some embodiments, one siRNA or sdRNA targets CISH and one siRNA or sdRNA targets TIM3. In some embodiments, one siRNA or sdRNA targets CISH and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets CISH and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets CISH and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets CBLB and one siRNA or sdRNA targets TIM3. In some embodiments, one sdRNA targets CD39 and one sdRNA targets CD69. In some embodiments, one siRNA or sdRNA targets CBLB and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets CBLB and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets CBLB and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets PD-1. In some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets LAG3. In some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets CBLB. In some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets CTLA-4. In some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets TIM3 and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets CTLA-4 and one siRNA or sdRNA targets TIGIT. In some embodiments, one siRNA or sdRNA targets CTLA-4 and one siRNA or sdRNA targets TET2. In some embodiments, one siRNA or sdRNA targets TIGIT and one siRNA or sdRNA targets TET2.

As discussed herein, embodiments of the present invention provide tumor infiltrating lymphocytes (TILs) that have been genetically modified by gene editing to enhance their therapeutic effects. Embodiments of the invention encompass gene editing via nucleotide insertion (RNA or DNA) into TIL populations to promote expression of one or more proteins and repress expression of one or more proteins and combinations thereof Embodiments of the invention also provide Methods for expanding TILs into a therapeutic population, wherein the methods comprise gene editing TILs. There are several gene editing techniques available for genetically modifying TIL populations that are suitable for use in accordance with the present invention. Such methods include those described below as well as the viral and transposon methods described elsewhere herein. In some embodiments, methods of genetically modifying TIL, MIL, or PBL to express CCR may also include modifications that inhibit gene expression by stably knocking out such genes or temporarily attenuating such genes.

In some embodiments, the method comprises a method of genetically modifying a population of TILs in the first population, the second population and/or the third population as described herein. In some embodiments, methods of genetically modifying a population of TILs include the step of stably incorporating genes for the production or repression (eg, silencing) of one or more proteins. In some embodiments, the method of genetically modifying a population of TILs includes the step of electroporation. Electroporation methods are known in the art and are described, for example, in Tsong, Biophysical Journal 1991, 60 , 297-306 and US Patent Application Publication No. 2014/0227237 A1, their respective disclosures The contents are incorporated herein by reference. Other electroporation methods known in the art, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, Nos. 5,304,120, 5,318,514, 6,010,613 and 6,078,490, the disclosures of which are incorporated herein by reference. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising applying a A series of at least three single, operator-controlled steps of independently programmed DC electrical pulses with field strengths equal to or greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two or three of the following characteristics (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different in pulse width from each other; and (3) the first group of the at least A first pulse interval of two of the three pulses is different from a second pulse interval of two of the second set of at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising applying a The step of a series of at least three single, operator-controlled independently programmed DC electrical pulses having a field strength equal to or greater than 100 V/cm, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising applying a The step of a series of at least three single, operator-controlled independently programmed DC electrical pulses having a field strength equal to or greater than 100 V/cm, wherein at least two of the at least three pulses differ from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or temporary changes in the TIL, comprising applying a A series of steps of at least three single, operator-controlled independently programmed DC electrical pulses having a field strength equal to or greater than 100 V/cm, wherein the first pulse interval of two of the at least three pulses in the first set is separated from the second The second pulse intervals of two of the set of at least three pulses are different. In some embodiments, the electroporation method is a pulsed electroporation method comprising the step of treating the TIL with a pulsed electric field to induce pore formation in the TIL, comprising the step of applying a series of at least three DC electrical pulses to the TIL at a field strength equal to or Greater than 100 V/cm, wherein the series of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) At least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the two of the at least three pulses in the first group and the interval between the two of the at least three pulses in the second group The second pulse interval was varied such that the induced pores lasted for a relatively long period of time and so that the viability of the TIL was maintained. In some embodiments, the method of genetically modifying a population of TILs comprises the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating and endocytosis) are known in the art and described in: Graham and van der Eb, Virology 1973, 52 , 456- 467; Wigler et al., Proceedings of the National Academy of Sciences USA 1979, 76 , 1373-1376; and Chen and Okayarea, Molecular Cell Biology 1987, 7 , 2745-2752; and US Patent No. 5,593,875, each of which The disclosure is incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs comprises the step of lipofection. Lipofectamine methods such as the use of cationic lipids N- [1-(2,3-dioleyloxy)propyl]-n ,n,n -trimethylammonium chloride (DOTMA) and dioleyl Methods for 1:1 (w/w) liposome formulation of phosphatidylethanolamine (DOPE) in filtered water are known in the art and described in: Rose et al., Biotechnology 1991, 10 , 520-525 and Felgner et al., Proceedings of the National Academy of Sciences, 1987, 84 , 7413-7417 and US Patent Nos. 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484 and 7,687,070 , the respective disclosures of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a population of TILs includes the step of transfection using the methods described in U.S. Pat. The disclosure is incorporated herein by reference. The TILs can be the first TIL population, the second TIL population, and/or the third TIL population as described herein.

According to one embodiment, the gene editing method may comprise the use of programmable nucleases that mediate double-stranded or single-stranded breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific gene body loci, i.e., they rely on recognizing a specific DNA sequence within the gene body to target the nuclease domain to this location and mediate Generates a double-stranded break at the target sequence. Double-stranded breaks in DNA then recruit endogenous repair mechanisms to the site of the break to mediate genome editing by non-homologous end joining (NHEJ) or homology-directed repair (HDR). Thus, repair of the break can result in the introduction of insertion/deletion mutations that disrupt (eg, silence, suppress, or enhance) the gene product of interest.

Major classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-related nucleases. Nucleases (eg CRISPR/Cas9). These nuclease systems can be roughly classified into two categories based on their DNA recognition modes: ZFNs and TALENs achieve specific DNA binding through protein-DNA interactions, while CRISPR systems, such as Cas9, RNA guides molecules and targets specific DNA sequences through protein-DNA interactions. See eg Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene editing methods that can be used in accordance with the TIL expansion methods of the present invention include CRISPR methods, TALE methods, and ZFN methods, which are described in more detail below. According to one embodiment, the method for expanding TILs into a therapeutic population can be according to any embodiment of the methods described herein (e.g., Gen 2) or as described in U.S. Patent Application Publication No. US 2020/0299644 A1 and US Pat. 2020/0121719 A1 and U.S. Patent No. 10,925,900, the disclosure of which is incorporated herein by reference, wherein the method further comprises one or more of CRISPR methods, TALE methods, or ZFN methods. At least a portion of the TILs are gene-edited to generate TILs that may enhance the therapeutic effect. According to one embodiment, gene editing can be assessed by comparing gene-edited TILs with unmodified TILs in vitro, e.g., by assessing in vitro effector functions, cytokine profiles, etc. compared to unmodified TILs Improved therapeutic effect of TIL. In certain embodiments, the methods comprise gene editing a population of TILs using CRISPR, TALE and/or ZFN methods.

In some embodiments of the invention, electroporation is used to deliver gene editing systems, such as CRISPR, TALEN and ZFN systems. In some embodiments of the invention, the electroporation system is a flow electroporation system. An example of a suitable flow electroporation system suitable for some embodiments of the invention is the commercially available MaxCyte STX system. There are several alternative commercially available electroporation instruments that may be suitable for use in the present invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Celelectricon), Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax) or siPORTer96 (Ambion). In some embodiments of the invention, the electroporation system forms a closed sterile system together with the rest of the TIL amplification method. In some embodiments of the invention In embodiments, the electroporation system is a pulsed electroporation system as described herein, and together with the remainder of the TIL expansion method forms a closed sterile system.

The method for expanding TILs into a therapeutic population can be according to any embodiment of the methods described herein (e.g. Gen 2) or as described in US Patent Application Publication Nos. US 2020/0299644 A1 and US 2020/0121719 A1 and as described in US Pat. No. 10,925,900, the disclosure of which is incorporated herein by reference, wherein the method further comprises gene editing at least a portion of the TILs by a CRISPR method (eg, CRISPR/Cas9 or CRISPR/Cpf1). According to certain embodiments, the use of CRISPR methods during the TIL expansion process results in silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of CRISPR methods during the TIL expansion process results in enhanced expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. Methods of gene editing using the CRISPR system are also referred to herein as CRISPR methods. There are three types of CRISPR systems that incorporate RNA and Cas proteins and can be used in accordance with the present invention: Type I, II and III. Type II CRISPR (exemplified by Cas9) is one of the best characterized systems.

CRISPR technology is adapted from the natural defense mechanisms of bacteria and archaea (the domain of single-celled microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins, including Cas9, to block the attack of viruses and other exosomes by shredding and destroying the DNA of foreign invaders. CRISPR is a specialized region of DNA with two unique features: the presence of nucleotide repeats and spacers. Repeats of nucleotides are distributed throughout the CRISPR region, with short foreign DNA segments (spacers) interspersed in the repeats. In type II CRISPR/Cas systems, spacers are integrated within the CRISPR gene body locus and are transcribed and processed into short CRISPR RNA (crRNA). These crRNAs anneal to transactivating crRNAs (tracrRNAs) and direct Cas proteins for sequence-specific cleavage and silencing of pathogenic DNA. Target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA binding region. Thus the CRISPR/Cas system can be retargeted to cleave almost any DNA sequence by redesigning crRNA. The crRNA and tracrRNA in the native system can be simplified to a single guide RNA (sgRNA) of about 100 nucleotides for genetic engineering. The CRISPR/Cas system can be directly carried into human cells by co-delivering plastids expressing the Cas9 endonuclease and essential crRNA components. Different Cas protein variants can be used to reduce targeting limitations (eg heterologs of Cas9 such as Cpf1).

Non-limiting examples of genes that can be silenced or suppressed by permanent gene editing of TILs using the CRISPR approach include CD39, CD69, PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-limiting examples of genes that can be enhanced by permanent gene editing of TILs via CRISPR methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, and IL-21.

Examples of systems, methods, and compositions that utilize the CRISPR approach to alter the expression of a gene sequence of interest and that can be used in accordance with embodiments of the present invention are described in U.S. Patent Nos. 8,697,359, 8,993,233, 8,795,965, 8,771,945, Nos. 8,889,356, 8,865,406, 8,999,641, 8,945,839, 8,932,814, 8,871,445, 8,906,616, and 8,895,308, the disclosures of each of which are incorporated herein by reference. Resources for performing CRISPR methods, such as plasmids for expressing CRISPR/Cas9 and CRISPR/Cpf1, are commercially available from companies such as GenScript.

In some embodiments, genetic modification of TIL populations as described herein can be performed using the CRISPR/Cpf1 system as described in US Pat. No. 9,790,490, the disclosure of which is incorporated herein by reference.

The method for expanding TILs into a therapeutic population can be according to any embodiment of the methods described herein (e.g. Gen 2) or as described in US Patent Application Publication Nos. US 2020/0299644 A1 and US 2020/0121719 A1 and as described in US Pat. No. 10,925,900, the disclosures of which are incorporated herein by reference, wherein the method further comprises gene editing at least a portion of the TILs by the TALE method. According to certain embodiments, the use of TALE methods during the TIL expansion process results in silencing or reduction in the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, use of the TALE approach during the TIL expansion process results in enhanced expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

TALE stands for "transcription activator-like effector" proteins, which include TALENs ("transcription activator-like effector nucleases"). Methods of gene editing using the TALE system are also referred to herein as TALE methods. TALEs are naturally occurring proteins from the plant pathogenic bacterium Xanthomonas and contain a DNA-binding domain composed of a series of repeat domains of 33-35 amino acids each recognizing a single base pair. TALE specificity is determined by two hypervariable amino acids called repeat-variable di-residues (RVDs). Modular TALE repeats are linked together to recognize contiguous DNA sequences. Specific RVDs in DNA-binding domains recognize bases in the locus of interest, providing structural features to assemble predictable DNA-binding domains. The DNA binding domain of the TALE was fused to the catalytic domain of the type IIS FokI endonuclease to generate a targetable TALE nuclease. To induce site-specific mutations, two individual TALEN arms separated by a 14-20 base pair spacer region bring FokI monomers closer together to dimerize and generate targeted double-stranded breaks.

Several large systematic studies using various assembly methods indicate that TALE repeats can be incorporated to recognize almost any user-defined sequence. Custom-designed TALE arrays were also provided by Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). )) commercially available. TALE and TALEN methods suitable for use in the present invention are described in US Patent Application Publication Nos. US 2011/0201118 A1, US 2013/0117869 A1, US 2013/0315884 A1, US 2015/0203871 A1 and US 2016/0120906 A1, the respective disclosures of which are incorporated herein by reference.

Non-limiting examples of genes that can be silenced or suppressed by permanent gene editing of TILs using the TALE approach include CD39, CD69, PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-limiting examples of genes that can be enhanced by permanent gene editing of TILs via the TALE approach include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, and IL-21.

Examples of systems, methods, and compositions that alter the expression of target gene sequences by the TALE approach and that can be used in accordance with embodiments of the present invention are described in US Patent No. 8,586,526, which is incorporated herein by reference.

The method for expanding TILs into a therapeutic population can be according to any embodiment of the methods described herein or as described in U.S. Patent Application Publication Nos. US 2020/0299644 A1 and US 2020/0121719 A1 and U.S. Patent No. 10,925,900, the disclosures of which are incorporated herein by reference, wherein the method further comprises gene editing at least a portion of the TILs by zinc finger or zinc finger nuclease methods. According to certain embodiments, the use of the zinc finger approach during the TIL expansion process results in silencing or reduction in the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, use of the zinc finger approach during the TIL expansion process results in enhanced expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

Individual zinc fingers in a conserved ββα configuration contain approximately 30 amino acids. Several amino acids on the surface of the α-helix usually contact 3 bp in the DNA major groove with varying levels of selectivity. Zinc fingers have two protein domains. The first domain is the DNA binding domain, which includes eukaryotic transcription factors and contains zinc fingers. The second domain is the nuclease domain, which includes the FokI restriction enzyme and is responsible for catalytic cleavage of DNA.

The DNA binding domain of an individual ZFN typically contains between three and six individual zinc finger repeats and each recognizes between 9 and 18 base pairs. Even a pair of 3-finger ZFNs recognizing a total of 18 base pairs could theoretically target a single locus in the mammalian genome if the zinc finger domains were specific for their intended target sites. One approach to generating new zinc finger arrays is to combine smaller zinc finger "modules" with known specificities. The most common modular assembly process involves combining three separate zinc fingers that each recognize a 3 base pair DNA sequence to generate a 3 finger array that recognizes a 9 base pair target site. Alternatively, selection-based methods, such as oligomerized pool engineering (OPEN), can be used to select new zinc finger arrays from randomly grouped libraries that account for context dependencies between adjacent fingers Sexual interaction (context-dependent interaction). Engineered zinc fingers are commercially available from Sangamo Biosciences (Richmond, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA).

Non-limiting examples of genes that can be silenced or suppressed by permanent gene editing of TILs using the zinc finger approach include CD39, CD69, PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish , TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6 , CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 , GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR.

Non-limiting examples of genes that can be enhanced via permanent gene editing of TILs via the zinc finger approach include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, and IL-21.

Examples of systems, methods, and compositions that alter the expression of target gene sequences by the zinc finger approach and that can be used in accordance with embodiments of the present invention are described in U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136號、第6,824,978號、第6,866,997號、第6,933,113號、第6,979,539號、第7,013,219號、第7,030,215號、第7,220,719號、第7,241,573號、第7,241,574號、第7,585,849號、第7,595,376號、第6,903,185號and No. 6,479,626, each of which is incorporated herein by reference.

Further examples of systems, methods and compositions that alter the expression of target gene sequences by means of a zinc finger approach and that can be used in accordance with other embodiments of the invention are described in Beane et al., Molecular Therapy, 2015, 23 , 1380-1390 , the disclosure of which is incorporated herein by reference.

In some embodiments, TILs are optionally genetically engineered to include additional functions including, but not limited to, high affinity TCRs, e.g., targeting tumor-associated antigens such as MAGE-1, HER2, or NY-ESO-1 or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (such as mesothelin) or lineage-restricted cell surface molecules (such as CD19). In some embodiments, genetic engineering can be used to gene edit TILs, including gene knockout of specific target genes, such as genes encoding PD-1 and CTLA-4 or CD39 and CD69. In certain embodiments, the method comprises genetically engineering the TIL population to include a high affinity TCR, e.g., a TCR targeting a tumor-associated antigen, such as MAGE-1, HER2, or NY-ESO-1; or a TCR associated with a tumor-associated cell Chimeric antigen receptors (CARs) bound by surface molecules such as mesothelin or lineage-restricted cell surface molecules such as CD19. Suitably, the population of TILs may be a first population, a second population and/or a third population as described herein. E. Closed system for TIL manufacturing

The present invention provides for the use of a closed system during the TIL culture procedure. Such closed systems allow the prevention and/or reduction of microbial contamination, the use of fewer culture bottles and the reduction of costs. In some embodiments, the closed system uses two containers.

Such containment systems are well known in the art and can be found, for example, at http://www.fda.gov/cber/guidelines.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779 .htm.

A sterile connecting device (STCD) creates a sterile weld between two compatible pieces of tubing. This procedure allows aseptic connection of multiple vessels and tube diameters. In some embodiments, the closure system includes a luer lock and heat sealing system as described in the Examples. In some embodiments, the closed system is accessed via a syringe under aseptic conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the Examples is used. In some embodiments, the TIL is formulated into a final product formulation vessel according to the methods described in the Examples herein.

In some embodiments, the closed system uses one container from the time the tumor fragments are obtained until the TIL is ready to be administered to a patient or cryopreserved. In some embodiments, when two containers are used, the first container is a closed G container, and the TIL population is centrifuged and transferred to an infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is an infusion bag containing HypoThermosol. A closed system or a closed TIL cell culture system is characterized in that once the tumor sample and/or tumor fragments have been added, the system is tightly sealed from the outside to form a closed environment free from contamination by bacteria, fungi and/or any other microorganisms.

In some embodiments, the reduction in microbial contamination is between about 5% and about 100%. In some embodiments, the reduction in microbial contamination is between about 5% and about 95%. In some embodiments, the reduction in microbial contamination is between about 5% and about 90%. In some embodiments, the reduction in microbial contamination is between about 10% and about 90%. In some embodiments, the reduction in microbial contamination is between about 15% and about 85%. In some embodiments, the reduction in microbial contamination is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, About 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100% %.

The closed system allows the growth of TILs in the absence and/or with significantly reduced microbial contamination.

In addition, the pH, partial pressure of carbon dioxide, and partial pressure of oxygen of the TIL cell culture environment each vary with cell culture. Therefore, even if the medium suitable for cell culture is circulated, the closed environment needs to be constantly maintained as an optimal environment for TIL proliferation. For this purpose, it is desirable to monitor the physical factors of pH, partial pressure of carbon dioxide and partial pressure of oxygen in the culture medium of the closed environment by means of sensors, the signals of which are used to control the gas installed at the entrance of the culture environment Exchanger, and real-time adjustment of the gas partial pressure of the closed environment according to the changes in the culture medium in order to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system that incorporates, at the inlet to the closed environment, a gas exchanger equipped with monitoring devices for measuring the pH, partial pressure of carbon dioxide, and partial pressure of oxygen of the closed environment, and by Optimizing the cell culture environment by automatically adjusting the gas concentration based on the signal from the monitoring device.

In some embodiments, the pressure within the closed environment is controlled continuously or intermittently. That is, the pressure in a closed environment can be varied by means of, for example, a pressure maintenance device, ensuring that the space is suitable for TIL growth under positive pressure conditions or promoting fluid leakage and thus cell proliferation under negative pressure conditions. Furthermore, by intermittently applying negative pressure, it is possible to uniformly and efficiently replace the circulating liquid in the closed environment by temporarily reducing the volume of the closed environment.

In some embodiments, optimal culture components for TIL proliferation can be replaced or added, and factors including, for example, IL-2 and/or OKT3 and combinations can be added. F. Cryopreservation of TIL selected according to the situation

The bulk TIL population (eg, the second TIL population) or the expanded TIL population (eg, the third TIL population) can optionally be cryopreserved. In some embodiments, cryopreservation occurs in a therapeutic TIL population. In some embodiments, cryopreservation occurs with TILs collected after the second expansion. In some embodiments, cryopreservation occurs as illustrated in FIG. 8 (such as, in particular, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) TIL in Step F. In some embodiments, the TILs are stored cryopreserved in an infusion bag. In some embodiments, the TILs are stored frozen prior to placement in the infusion bag. In some embodiments, TILs are stored cryopreserved and not placed in an infusion bag. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethylsulfoxide (DMSO). This is typically done by placing the TIL population in a freezing solution such as 85% complement inactivated AB serum and 15% dimethylsulfoxide (DMSO). Cells in solution were placed in cryogenic vials and stored at -80°C for 24 hours, optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, cells were removed from the freezer and thawed in a 37°C water bath until approximately 4/5 of the solution was thawed. Cells are generally resuspended in complete medium and washed one or more times as appropriate. In some embodiments, thawed TILs can be calculated and assessed for viability as known in the art.

In some embodiments, TIL populations are cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLife Solutions). In some embodiments, the TIL population is cryopreserved using a cryopreservation medium containing dimethylsulfoxide (DMSO). In some embodiments, TIL populations are cryopreserved using a 1:1 (vol:vol) ratio of CS10 to cell culture medium. In some embodiments, TIL populations are cryopreserved using about a 1:1 (vol:vol) ratio of CS10 to cell culture medium (further comprising additional IL-2).

As discussed above and as shown in FIG. 1 and/or FIG. 8 (in particular such as FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) As exemplified in Steps A through E provided in , cryopreservation can occur at various time points in the TIL expansion procedure. In some embodiments, the amplified TIL population after the first amplification (as provided, for example, according to step B) or in accordance with FIG. 1 or FIG. 8 (especially, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) the expanded TIL population after one or more second amplifications of step D) may be cryopreserved. Cryopreservation can generally be accomplished by placing TIL populations in a freezing solution such as 85% complement-deactivated AB serum and 15% dimethylsulfoxide (DMSO). Cells in solution were placed in cryogenic vials and stored at -80°C for 24 hours, optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013 , 52 , 978-986. In some embodiments, TILs are stored cryopreserved in 5% DMSO. In some embodiments, TILs are cryopreserved in cell culture medium plus 5% DMSO. In some embodiments, TILs are cryopreserved according to the methods provided in Example 6.

When appropriate, cells were removed from the freezer and thawed in a 37°C water bath until approximately 4/5 of the solution was thawed. Cells are generally resuspended in complete medium and washed one or more times as appropriate. In some embodiments, thawed TILs can be calculated and assessed for viability as known in the art.

In some cases, from FIG. 1 or FIG. 8 (specifically, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) In Step B, the TIL population can be immediately cryopreserved using the protocol discussed below. Alternatively, the TIL population of the subject may be subjected to a TIL from FIG. 1 or FIG. 8 (specifically, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8G) step C and step D, and then from FIG. 1 or FIG. 8 (specifically, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8F and/or step D in FIG. 8G) and then cryopreserved. Similarly, where genetically modified TILs are to be used in therapy, from FIG. 1 or FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) step B or step D, the TIL population can be genetically modified for suitable processing. G. Phenotypic Characterization of Expanded TILs

In some embodiments, TILs are analyzed after expansion for expression of various phenotypic markers, including those described herein and in the Examples. In some embodiments, the expression of one or more phenotypic markers is examined. In some embodiments, from FIG. 1 or FIG. 8 (specifically, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. Phenotypic characteristics of TILs were analyzed after the first amplification in step B of 8G). In some embodiments, from FIG. 1 or FIG. 8 (specifically, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. Phenotypic characteristics of TILs were analyzed during transition in step C of 8G). In some embodiments, based on information from FIG. 1 or FIG. 8 (specifically, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or Phenotypic characteristics of TILs were analyzed during transition of step C of FIG. 8G ) and after cryopreservation. In some embodiments, based on information from FIG. 1 or FIG. 8 (specifically, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or TILs were analyzed for phenotypic characteristics after the second amplification of step D of FIG. 8G ). In some embodiments, based on information from FIG. 1 or FIG. 8 (specifically, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or TILs were analyzed for phenotypic characteristics after two or more amplifications of step D of FIG. 8G ).

In some embodiments, the marker is selected from the group consisting of CD8 and CD28. In some embodiments, the expression of CD8 is examined. In some embodiments, the expression of CD28 is examined. In some embodiments, compared to other procedures (such as FIG. 8 (in particular, such as FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8G) provided in the Gen 3 program), compared to, for example, Fig. 8 (in particular, such as Fig. 8A and/or Fig. 8B and/or Fig. 8C and/or Fig. 8D and/or Fig. 8E and/or Fig. 8F and/or or the 2A procedure provided in FIG. 8G ), the expression levels of CD8 and/or CD28 of TILs generated according to the procedure of the present invention are relatively high. In some embodiments, compared to other programs (such as the Gen 3 program provided in FIG. 8 (especially, for example, FIG. 8B ), compared to, for example, FIG. Or the 2A procedure provided in FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ), the CD8 expression level of TIL generated according to the procedure of the present invention is higher. In some embodiments, compared to other procedures (such as FIG. 8 (in particular, such as FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8G)) TILs generated according to the procedure of the present invention have a higher expression level of CD28 compared to the 2A procedure as provided, for example, in FIG. In some embodiments, high CD28 expression is indicative of a younger, more persistent TIL phenotype. In some embodiments, the expression of one or more regulatory markers is measured.

In some embodiments, during any step of the methods for expanding tumor infiltrating lymphocytes (TILs) described herein, the first TIL population, the second TIL population, the The third TIL population or the collected TIL population.

In some embodiments, compared to other procedures (such as FIG. 8 (in particular, such as FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. The Gen 3 program presented in 8G) has a higher percentage of central memory cells for TILs produced according to the program of the invention compared to the program 2A as provided eg in FIG. 8 (in particular eg FIG. 8A ). In some embodiments, the memory markers of central memory cells are selected from the group consisting of CCR7 and CD62L.

In some embodiments, CD4+ and/or CD8+ TIL memory subsets can be divided into different memory subsets. In some embodiments, the CD4+ and/or CD8+ TILs comprise naive (CD45RA+CD62L+) TILs. In some embodiments, the CD4+ and/or CD8+ TILs comprise central memory (CM; CD45RA-CD62L+) TILs. In some embodiments, the CD4+ and/or CD8+ TILs comprise effector memory (EM; CD45RA-CD62L-) TILs. In some embodiments, the CD4+ and/or CD8+ TILs comprise RA+ effector memory/effector (TEMRA/TEFF; CD45RA+CD62L+) TILs.

In some embodiments, the TIL expresses one or more markers selected from the group consisting of granzyme B, perforin, and granlysin. In some embodiments, the TIL expresses granzyme B. In some embodiments, the TIL expresses a perforin. In some embodiments, the TIL expresses granulysin.

In some embodiments, interleukin release from restimulated TILs can also be assessed using an interleukin release assay. In some embodiments, TILs can be assessed for interferon-gamma (IFN-gamma) secretion. In some embodiments, IFN-γ secretion is measured by ELISA assay. In some embodiments, IFN-γ is secreted after a rapid second amplification step, as in, for example, Figure 8 (in particular, for example, Figure 8A and/or Figure 8B and/or Figure 8C and/or Figure 8D and/or Figure 8C and/or Figure 8D and/or Figure 8). 8E and/or FIG. 8F and/or FIG. 8G ) provided after step D is measured by ELISA analysis. In some embodiments, TIL health is measured by IFN-γ (IFN-γ) secretion. In some embodiments, IFN-γ secretion is indicative of active TIL. In some embodiments, potency assays for IFN-γ production are employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by measuring the content of interleukin IFN-γ in TIL medium stimulated with antibodies against CD3, CD28 and CD137/4-1BB. The IFN-γ content in the medium from these stimulated TILs can be determined by measuring IFN-γ release. In some embodiments, provided in, for example, FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) The IFN-γ production of Step D TILs in the Gen 3 program was increased compared to Step D in the 2A program as provided for example in Figure 8 (especially eg Figure 8A), indicating an increased cytotoxic potential of Step D TILs. In some embodiments, IFN-γ secretion is doubled, doubled, tripled, quadrupled, or fivefold or more increased. In some embodiments, IFN-γ secretion is doubled. In some embodiments, IFN-γ secretion is increased two-fold. In some embodiments, the secretion of IFN-γ is increased three-fold. In some embodiments, IFN-γ secretion is increased four-fold. In some embodiments, IFN-γ secretion is increased five-fold. In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ is measured in ex vivo TILs. In some embodiments, IFN-γ is measured ex vivo in TILs, including TILs produced by methods of the invention including, for example, the method of FIG. 8B .

In some embodiments, TILs capable of secreting at least one-fold, two-fold, three-fold, four-fold or five-fold or more IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B ). and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least one-fold more IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least two-fold more IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least three times more IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least four times more IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least five times more IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs.

In some embodiments, TILs capable of secreting at least 100 pg/mL to about 1000 pg/mL or more of IFN-γ are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C ). and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TILs. In some embodiments, capable of secreting at least 200 pg/mL, at least 250 pg/mL, at least 300 pg/mL, at least 350 pg/mL, at least 400 pg/mL, at least 450 pg/mL, at least 500 pg/mL, At least 550 pg/mL, at least 600 pg/mL, at least 650 pg/mL, at least 700 pg/mL, at least 750 pg/mL, at least 800 pg/mL, at least 850 pg/mL, at least 900 pg/mL, at least 950 pg/mL TILs of pg/mL or at least 1000 pg/mL or more of IFN-γ are obtained by the amplification method of the present invention (including, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 200 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 200 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 300 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 400 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 500 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 600 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 700 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 800 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 900 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 1000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 2000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 3000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 4000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 5000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 6000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 7000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 8000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 9000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 10.000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 15.000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 20.000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 25.000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 30.000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 35.000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 40.000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 45.000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs. In some embodiments, TILs capable of secreting at least 50.000 pg/mL IFN-γ are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) generated TILs.

In some embodiments, TILs capable of secreting at least 100 pg/mL/5e5 cells to about 1000 pg/mL/5e5 cells or more IFN-γ are expanded by methods of the invention (including, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, capable of secreting at least 200 pg/mL/5e5 cells, at least 250 pg/mL/5e5 cells, at least 300 pg/mL/5e5 cells, at least 350 pg/mL/5e5 cells, at least 400 pg/mL/5e5 cells pg/mL/5e5 cells, at least 450 pg/mL/5e5 cells, at least 500 pg/mL/5e5 cells, at least 550 pg/mL/5e5 cells, at least 600 pg/mL/5e5 cells, at least 650 pg/mL/5e5 cells, at least 700 pg/mL/5e5 cells, at least 750 pg/mL/5e5 cells, at least 800 pg/mL/5e5 cells, at least 850 pg/mL/5e5 cells, at least 900 pg/mL/5e5 cells TILs of pg/mL/5e5 cells, at least 950 pg/mL/5e5 cells, or at least 1000 pg/mL/5e5 cells or more of IFN-γ were expanded by the methods of the present invention (including, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 300 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 400 pg/mL/5e5 cells are obtained by expansion methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 500 pg/mL/5e5 cells are obtained by expansion methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 600 pg/mL/5e5 cells are obtained by expansion methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 700 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 800 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 900 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 1000 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 2000 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 3000 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 4000 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 5000 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 6000 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 7000 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 8000 pg/mL/5e5 cells are expanded by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 9000 pg/mL/5e5 cells are expanded by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 10.000 pg/mL/5e5 cells are expanded by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 15.000 pg/mL/5e5 cells are expanded by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 20.000 pg/mL/5e5 cells are expanded by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 25.000 pg/mL/5e5 cells are expanded by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 30.000 pg/mL/5e5 cells are expanded by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 35.000 pg/mL/5e5 cells are expanded by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 40.000 pg/mL/5e5 cells are obtained by expansion methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 45.000 pg/mL/5e5 cells are expanded by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 50.000 pg/mL/5e5 cells are obtained by expansion methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G method) generated TIL.

The diverse antigen receptors of T and B lymphocytes are generated by somatic recombination of a limited but large number of gene segments. These gene segments: V (variable region), D (diversity region), J (junction region) and C (constant region) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCR). The present invention provides a method for generating TILs that exhibit and increase the diversity of the T cell repertoire. In some embodiments, TILs obtained by the methods of the invention exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained by the methods of the invention exhibit increased T cell repertoire diversity compared to freshly collected TILs and/or TILs prepared using methods other than those provided herein, such other The method includes, for example, a method other than that embodied in FIG. 8 (in particular, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) . In some embodiments, TILs obtained by the methods of the invention exhibit T cells compared to freshly collected TILs and/or TILs prepared using a method called Gen 2 as exemplified in FIG. 8 (in particular, for example, FIG. 8A ). Repository diversity increased. In some embodiments, the TILs obtained in the first expansion exhibit increased T cell repertoire diversity. In some embodiments, increasing diversity is increasing immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, present in immunoglobulin heavy chains. In some embodiments, the diversity is present in immunoglobulins, in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and/or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, TCRab (ie, TCRα/β) expression is increased. In some embodiments, a program as described herein (e.g., a Gen 3 program) exhibits a higher diversity of colonies based on the number of unique peptide CDRs within a sample compared to other programs, such as a program called Gen 2 .

In some embodiments, activation and depletion of TILs can be determined by examining one or more markers. In some embodiments, activation and depletion can be determined using multicolor flow cytometry. In some embodiments, activation and depletion of markers includes, but is not limited to, one or more markers selected from the group consisting of: CD39, CD69, CD3, PD-1, 2B4/CD244, CD8, CD25 , BTLA, KLRG, TIM-3, CD194/CCR4, CD4, TIGIT, CD183, CD69, CD95, CD127, CD103 and/or LAG-3. In some embodiments, activation and depletion of markers includes, but is not limited to, one or more markers selected from the group consisting of: CD39, CD69, BTLA, CTLA-4, ICOS, Ki67, LAG-3 , PD-1, TIGIT and/or TIM-3. In some embodiments, activation and depletion of markers includes, but is not limited to, one or more markers selected from the group consisting of: CD39, CD69, BTLA, CTLA-4, ICOS, Ki67, LAG-3 , CD103+/CD69+, CD103+/CD69-, PD-1, TIGIT and/or TIM-3. In some embodiments, T cell markers, including activation and depletion markers, can be determined and/or analyzed to examine T cell activation, suppression or function. In some embodiments, T cell markers may include, but are not limited to, one or more markers selected from the group consisting of: CD39, CD69, TIGIT, CD3, FoxP3, Tim-3, PD-1, CD103 , CTLA-4, LAG-3, BTLA-4, ICOS, Ki67, CD8, CD25, CD45, CD4 and/or CD59.

In some embodiments, TILs exhibiting secretion of greater than 3000 pg/106 TILs to 300,000 pg/106 TILs or more of granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, exhibiting secretion of greater than 3000 pg/ ¹⁰⁶ TILs, greater than 5000 pg/ ¹⁰⁶ TILs, greater than 7000 pg/ ¹⁰⁶ TILs, greater than 9000 pg/ ¹⁰⁶ TILs, greater than 11000 pg/ ¹⁰⁶ TIL, above 13000 pg/ ¹⁰⁶ TIL, above 15000 pg/ ¹⁰⁶ TIL, above 17000 pg/ ¹⁰⁶ TIL, above 19000 pg/ ¹⁰⁶ TIL, above 20,000 pg/10 ⁶ TILs, higher than 40,000 pg/10 ⁶ TILs, higher than 60,000 pg/10 ⁶ TILs, higher than 80,000 pg/10 ⁶ TILs, higher than 100,000 pg/10 ⁶ TILs, higher than 120,000 pg/10 ⁶ TILs, above 140,000 pg/10 ⁶ TILs, above 160,000 pg/10 ⁶ TILs, above 180,000 pg/10 ⁶ TILs, above 200,000 pg/10 ⁶ TILs, above 220,000 pg/10 ⁶ TILs, above 240,000 pg/10 ⁶ TILs, above 260,000 pg/10 ⁶ TILs, above 280,000 pg/10 ⁶ TILs, above 300,000 pg/10 ⁶ TILs or more TILs of granzyme B are produced by the amplification method of the present invention (including, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) The TIL. In some embodiments, TILs exhibiting secretion of greater than ³⁰⁰⁰ pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than ⁵⁰⁰⁰ pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than 7000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising ^, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than 9000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising ^, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than 11000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising ^, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 13000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 15,000 pg/10 TIL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 17000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 19000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 20,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 40,000 pg/10 TIL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than ^60,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 80,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 100,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 120,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 140,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 160,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 180,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 200,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 220,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 240,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 260,000 pg/10 TIL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 280,000 pg/10 TIL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 300,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than 3000 pg/ ¹⁰⁶ TILs to 300000 pg/ ¹⁰⁶ TILs or more of granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, exhibiting secretion of greater than 3000 pg/ ¹⁰⁶ TILs, greater than 5000 pg/ ¹⁰⁶ TILs, greater than 7000 pg/ ¹⁰⁶ TILs, greater than 9000 pg/ ¹⁰⁶ TILs, greater than 11000 pg/ ¹⁰⁶ TIL, above 13000 pg/ ¹⁰⁶ TIL, above 15000 pg/ ¹⁰⁶ TIL, above 17000 pg/ ¹⁰⁶ TIL, above 19000 pg/ ¹⁰⁶ TIL, above 20,000 pg/10 ⁶ TILs, higher than 40,000 pg/10 ⁶ TILs, higher than 60,000 pg/10 ⁶ TILs, higher than 80,000 pg/10 ⁶ TILs, higher than 100,000 pg/10 ⁶ TILs, higher than 120,000 pg/10 ⁶ TILs, above 140,000 pg/10 ⁶ TILs, above 160,000 pg/10 ⁶ TILs, above 180,000 pg/10 ⁶ TILs, above 200,000 pg/10 ⁶ TILs, above 220,000 pg/10 ⁶ TILs, above 240,000 pg/10 ⁶ TILs, above 260,000 pg/10 ⁶ TILs, above 280,000 pg/10 ⁶ TILs, above 300,000 pg/10 ⁶ TILs or more TILs of granzyme B are produced by the amplification method of the present invention (including, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ) The TIL. In some embodiments, TILs exhibiting secretion of greater than ³⁰⁰⁰ pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than ⁵⁰⁰⁰ pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than ⁷⁰⁰⁰ pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than 9000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising ^, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than 11000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising ^, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 13000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 15,000 pg/10 TIL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 17000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 19000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 20,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than ^40,000 pg/10 TIL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater than ^60,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 80,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 100,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 120,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 140,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 160,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 180,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 200,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 220,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 240,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 260,000 pg/10 TIL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 280,000 pg/10 TIL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, TILs exhibiting secretion of greater ^than 300,000 pg/10 TIL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs.

In some embodiments, TILs exhibiting secretion of greater than 1000 pg/mL to 300,000 pg/mL or more of granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C ). and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G) generated TILs. In some embodiments, exhibits secretion greater than 1000 pg/mL, greater than 2000 pg/mL, greater than 3000 pg/mL, greater than 4000 pg/mL, greater than 5000 pg/mL, greater than 6000 pg/mL, Bigger TIL. Greater than 7000 pg/mL, greater than 8000 pg/mL, greater than 9000 pg/mL, greater than 10000 pg/mL, greater than 20000 pg/mL, greater than 30000 pg/mL, greater than 40000 pg/mL, greater than TILs of 50,000 pg/mL, greater than 60,000 pg/mL, greater than 70,000 pg/mL, greater than 80,000 pg/mL, greater than 90,000 pg/mL, greater than 100,000 pg/mL or more of granzyme B were obtained by this TILs generated by the inventive amplification method (including, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ). In some embodiments, TILs exhibiting secretion of greater than 1000 pg/mL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 2000 pg/mL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 3000 pg/mL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 4000 pg/mL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 5000 pg/mL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 6000 pg/mL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 7000 pg/mL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 8000 pg/mL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 9000 pg/mL of granzyme B are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 10,000 pg/mL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 20,000 pg/mL of granzyme B are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 30,000 pg/mL of granzyme B are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 40,000 pg/mL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 50,000 pg/mL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 60,000 pg/mL granzyme B are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 70,000 pg/mL granzyme B are amplified by the methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 80,000 pg/mL granzyme B are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 90,000 pg/mL granzyme B are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 100,000 pg/mL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 120,000 pg/mL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 140,000 pg/mL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 160,000 pg/mL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 180,000 pg/mL granzyme B are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 200,000 pg/mL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 220,000 pg/mL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 240,000 pg/mL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 260,000 pg/mL granzyme B are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 280,000 pg/mL of granzyme B are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL. In some embodiments, TILs exhibiting secretion of greater than 300,000 pg/mL granzyme B are amplified by methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or Figure 8E and/or Figure 8F and/or Figure 8G) generated TIL.

In some embodiments, the amplification methods of the invention generate expanded TIL populations that exhibit increased granzyme B secretion in vitro compared to non-expanded TIL populations, including, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or the TIL provided in FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G. In some embodiments, the expanded TIL population of the invention has at least one-fold to fifty-fold or more increased granzyme B secretion compared to a non-expanded TIL population. In some embodiments, IFN-γ secretion is at least one-fold, at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least eight-fold increased compared to a non-expanded TIL population times, at least nine times, at least ten times, at least twenty times, at least thirty times, at least forty times, at least fifty times or more. In some embodiments, granzyme B secretion is at least doubled in an expanded TIL population of the invention compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least a two-fold increase in granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least a three-fold increase in granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least a four-fold increase in granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least a five-fold increase in granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least a six-fold increase in granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least a seven-fold increase in granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least an eight-fold increase in granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least a nine-fold increase in granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least ten-fold increased granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least a twenty-fold increase in granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least thirty-fold increased granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least forty-fold increased granzyme B secretion compared to a non-expanded TIL population. In some embodiments, the expanded TIL population of the invention has at least a fifty-fold increase in granzyme B secretion compared to a non-expanded TIL population.

In some embodiments, TILs capable of secreting TNF-alpha (i.e., TNF-alpha) amounts that are at least one-fold, two-fold, three-fold, four-fold, or five-fold or more lower than IFN-γ secretion are TILs produced by the amplification method of the present invention (including, for example, the method of FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method). In some embodiments, TILs capable of secreting an amount of TNF-α that is at least one-fold lower than the amount of IFN-γ secreted are obtained by the amplification methods of the present invention (including, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least two-fold lower amounts of TNF-α than IFN-γ are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting an amount of TNF-α that is at least three times lower than the amount of IFN-γ secreted by the amplification methods of the invention (including, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least four-fold lower amounts of TNF-α than IFN-γ are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least five-fold lower amounts of TNF-alpha than IFN-gamma are obtained by amplification methods of the invention (comprising, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL.

In some embodiments, TILs capable of secreting at least 200 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more TNF-α (i.e., TNF-alpha) are amplified by the present invention TILs generated by methods (including, for example, the method of FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method). In some embodiments, TILs capable of secreting at least 500 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are expanded by methods of the invention (comprising, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 1000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are expanded by methods of the invention (including, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 2000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are expanded by methods of the invention (comprising, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 3000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are expanded by methods of the invention (comprising, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 4000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are expanded by methods of the invention (comprising, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 5000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are expanded by methods of the invention (comprising, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 6000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are expanded by the methods of the invention (including, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 7000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are expanded by methods of the invention (comprising, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 8000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are expanded by methods of the invention (comprising, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL. In some embodiments, TILs capable of secreting at least 9000 pg/mL/5e5 cells to about 10,000 pg/mL/5e5 cells or more of TNF-α are expanded by methods of the invention (comprising, for example, FIG. 8A and/or Or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G method) generated TIL.

In some embodiments, the content of IFN-γ and granzyme B is measured to determine the amplification method of the present invention (including, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) to characterize the phenotypes of TILs produced. In some embodiments, the content of IFN-γ and TNF-α is measured to determine the amplification method of the present invention (including, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) to characterize the phenotypes of TILs produced. In some embodiments, the content of granzyme B and TNF-α is measured to determine whether the amplification method of the present invention (including, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or Figure 8F and/or Figure 8G methods) to characterize the phenotypes of TILs produced. In some embodiments, the content of IFN-γ, granzyme B and TNF-α is measured to determine the amplification method of the present invention (including, for example, FIG. 8A and/or FIG. 8B and/or FIG. 8C and/or FIG. 8D and /or Figure 8E and/or Figure 8F and/or Figure 8G methods) phenotypic characteristics of TILs produced.

In some embodiments, phenotypic characteristics are examined after cryopreservation. H. Additional Process Embodiments

In some embodiments, the present invention provides methods for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) forming a plurality of tumors by processing a tumor sample obtained from an individual fragment to obtain a first TIL population derived from a tumor resected from an individual and to isolate a CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population from the first TIL population; (b) by including IL-2 and OKT -3 culture of CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL populations in cell culture medium for initial first expansion, wherein initial first expansion is performed for about 1 to 7 days or about 1 to 8 days Obtaining a second TIL population, wherein the number of the second TIL population is greater than that of the first TIL population; (c) by making the second TIL population and comprising IL-2, OKT-3 and exogenous antigen presenting cells (APC) Contacting the cell culture medium for rapid second expansion to produce a third TIL population, wherein the rapid second expansion is performed for about 1 to 11 days or about 1 to 10 days to obtain the third TIL population, wherein the third TIL population is therapeutic and (d) collecting the therapeutic TIL population obtained from step (c). In some embodiments, the step of rapid second expansion is divided into multiple steps to achieve vertical scale-up of culture by: (1) by using The small-scale culture grows the second TIL population for a period of about 3 to 4 days or about 2 to 4 days to perform a rapid second expansion; and then (2) effectuates transfer of the second TIL population from the small-scale culture to In a second container (e.g., a G-REX 500MCS container) larger than the first container, wherein in the second container, a second TIL population from a small-scale culture is cultivated in a larger-scale culture for about 4 to 7 days or a period of about 4 to 8 days. In some embodiments, the step of rapid expansion is divided into multiple steps to achieve lateral expansion of the culture scale in the following manner: (1) by using the first container (such as a G-REX 100MCS container) The small-scale culture grows the second TIL population for a period of about 3 to 4 days to perform a rapid second expansion; and then (2) achieves the transfer and distribution of the second TIL population from the first small-scale culture to at least 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers equal in size to the first container, wherein in each second container, the portion of the second TIL population from the first small-scale culture transferred to such second container is cultured in the second small-scale culture for a period of about 4 to 7 days or about 4 to 8 days . In some embodiments, the step of rapid expansion is divided into multiple steps to achieve horizontal expansion and vertical expansion of the culture scale in the following ways: (1) by using the first container (such as G-REX 100MCS container) performing a rapid second expansion by culturing the second population of TILs in small-scale cultures for a period of about 3 to 4 days, or about 2 to 4 days; and then (2) effecting the transfer of the second TILs from the first small-scale culture Populations are transferred and distributed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 sizes larger than the first container In larger second vessels (e.g., G-REX 500MCS vessels), wherein in each second vessel, the portion of the second TIL population transferred from the small-scale culture to such second vessel is grown as a larger-scale culture for approximately A period of 4 to 7 days or about 4 to 8 days. In some embodiments, the rapid expansion step is divided into multiple steps to achieve culture scale-up and scale-up by: (1) by using small A rapid second expansion of the second TIL population is grown in the large-scale culture over a period of about 3 to 4 days; and then (2) effecting transfer and distribution of the second TIL population from the first small-scale culture to at least 2, 3, or 4 Among the second containers (such as G-REX 500MCS containers) larger in size than the first container, wherein in each second container, the fraction of the second TIL population transferred from the small-scale culture to such second container is in the larger In large-scale culture, it is cultivated for a period of about 5 days to 7 days.

In some embodiments, the present invention provides methods for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) forming a plurality of tumors by processing a tumor sample obtained from an individual fragment to obtain a first TIL population derived from a tumor resected from an individual and to isolate a CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population from the first TIL population; (b) by including IL-2 and OKT -3 CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL populations are cultured in cell culture medium to initiate the first expansion, wherein the initial first expansion is carried out for about 1 to 8 days to obtain the second TIL population , wherein the second population of TILs is greater in number than the first population of TILs; (c) rapid TIL population by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and exogenous antigen-presenting cells (APCs). second expansion to produce a third population of TILs, wherein the rapid second expansion is performed for about 1 to 8 days to obtain a third population of TILs, wherein the third population of TILs is a population of therapeutic TILs; and (d) collected from step ( c) Obtained therapeutic TIL population. In some embodiments, the step of rapid second expansion is divided into multiple steps to achieve vertical scale-up of culture by: (1) by small scale in the first vessel (eg, G-REX 100MCS vessel) The second TIL population is grown in culture for a period of about 2 to 4 days for a rapid second expansion; and then (2) effecting transfer of the second TIL population from the small-scale culture to a second vessel larger than the first vessel (e.g. G-REX 500MCS container), where in the second container, the second TIL population from the small scale culture is cultured in the larger scale culture for a period of about 4 to 8 days. In some embodiments, the rapid expansion step is divided into multiple steps to achieve culture scale-up by: (1) by first small-scale culture in the first vessel (eg, G-REX 100MCS vessel) Medium-culturing the second TIL population for a period of about 2 to 4 days for a rapid second expansion; and then (2) effecting transfer and distribution of the second TIL population from the first mini-culture to at least 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers equal in size to the first container, wherein each second container In this, the portion of the second TIL population from the first mini-culture transferred to such second container is cultured in the second mini-culture for a period of about 4 to 6 days. In some embodiments, the rapid expansion step is divided into multiple steps to achieve culture scale-up and scale-up by: (1) by using small The second TIL population is grown in the scale culture for a period of about 2 to 4 days for a rapid second expansion; and then (2) effecting the transfer and distribution of the second TIL population from the first small scale culture to at least 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers larger in size than the first container (eg G-REX 500MCS container ), wherein in each second vessel, the portion of the second TIL population transferred from the small scale culture to such second vessel is cultured in the larger scale culture for a period of about 4 to 6 days. In some embodiments, the rapid expansion step is divided into multiple steps to achieve culture scale-up and scale-up by: (1) by using small A rapid second expansion of the second TIL population is grown in the large-scale culture over a period of about 3 to 4 days; and then (2) effecting transfer and distribution of the second TIL population from the first small-scale culture to at least 2, 3, or 4 Among the second containers (such as G-REX 500MCS containers) larger in size than the first container, wherein in each second container, the fraction of the second TIL population transferred from the small-scale culture to such second container is in the larger It is cultured for a period of about 4 to 5 days in scale culture.

In some embodiments, the present invention provides methods for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) forming a plurality of tumors by processing a tumor sample obtained from an individual fragment to obtain a first TIL population derived from a tumor resected from an individual and to isolate a CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population from the first TIL population; (b) by including IL-2 and OKT -3 culture of CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL populations in cell culture medium to initiate the first expansion, wherein the initial first expansion is carried out for about 1 to 7 days to obtain the second TIL population , wherein the second population of TILs is greater in number than the first population of TILs; (c) rapid TIL population by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and exogenous antigen-presenting cells (APCs). second expansion to produce a third population of TILs, wherein the rapid second expansion is performed for about 1 to 11 days to obtain a third population of TILs, wherein the third population of TILs is a population of therapeutic TILs; and (d) collected from step ( c) Obtained therapeutic TIL population. In some embodiments, the step of rapid second expansion is divided into multiple steps to achieve vertical scale-up of culture by: (1) by small scale in the first vessel (eg, G-REX 100MCS vessel) The second TIL population is grown for a period of about 3 to 4 days in culture for a rapid second expansion; and then (2) effecting transfer of the second TIL population from the small scale culture to a second vessel larger than the first vessel (e.g. G-REX 500MCS container), where in the second container, the second TIL population from the small scale culture is cultured in the larger scale culture for a period of about 4 to 7 days. In some embodiments, the rapid expansion step is divided into multiple steps to achieve culture scale-up by: (1) by first small-scale culture in the first vessel (eg, G-REX 100MCS vessel) A rapid second expansion is performed over a period of approximately 3 to 4 days in culturing the second TIL population; and then (2) effectuating the transfer and distribution of the second TIL population from the first mini-culture to at least 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers equal in size to the first container, wherein each second container In this, the portion of the second TIL population from the first mini-culture transferred to such second container is cultured in the second mini-culture for a period of about 4 to 7 days. In some embodiments, the rapid expansion step is divided into multiple steps to achieve culture scale-up and scale-up by: (1) by using small A rapid second expansion of the second TIL population is grown in the large-scale culture over a period of about 3 to 4 days; and then (2) effecting the transfer and distribution of the second TIL population from the first small-scale culture to at least 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers larger in size than the first container (eg G-REX 500MCS container ), wherein in each second vessel, the portion of the second TIL population transferred from the small scale culture to such second vessel is cultured in the larger scale culture for a period of about 4 to 7 days. In some embodiments, the step of rapid expansion is divided into multiple steps to achieve horizontal scale-up and vertical scale-up of culture by: (1) by adding A rapid second expansion is performed in the mini-culture over a period of about 4 days culturing the second TIL population; and then (2) effecting the transfer and distribution of the second TIL population from the first mini-culture to at least 2, Among 3 or 4 second containers (such as G-REX-g500MCS containers) larger in size than the first container, wherein in each second container, the second Portions of the TIL population were grown in larger scale cultures for a period of about 5 days.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that in step (b), the primary first amplification is by making the TILs double negative for CD39/CD69 and and/or the CD39 ^LO /CD69 ^LO population is performed in contact with a culture medium further comprising exogenous antigen-presenting cells (APCs), wherein the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b) number.

In other embodiments, the invention provides a modification of the method described in any preceding paragraph as applicable above, wherein in step (c), the culture medium is supplemented with additional exogenous APC.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 1.1:1 to exactly or about 20:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 1.1:1 to exactly or about 10:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 1.1:1 to exactly or about 9:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 1.1:1 to exactly or about 8:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 1.1:1 to exactly or about 7:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 1.1:1 to exactly or about 6:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or about 1.1:1 to exactly or about 5:1 range.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 1.1:1 to exactly or about 4:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 1.1:1 to exactly or about 3:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 1.1:1 to exactly or about 2.9:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 1.1:1 to exactly or about 2.8:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 1.1:1 to exactly or about 2.7:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 1.1:1 to exactly or about 2.6:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 1.1:1 to exactly or about 2.5:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 1.1:1 to exactly or about 2.4:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 1.1:1 to exactly or about 2.3:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 1.1:1 to exactly or about 2.2:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 1.1:1 to exactly or about 2.1:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 1.1:1 to exactly or about 2:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 2:1 to exactly or about 10:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 2:1 to exactly or about 5:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 2:1 to exactly or about 4:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 2:1 to exactly or about 3:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 2:1 to exactly or about 2.9:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 2:1 to exactly or about 2.8:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 2:1 to exactly or about 2.7:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 2:1 to exactly or about 2.6:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 2:1 to exactly or about 2.5:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 2:1 to exactly or about 2.4:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 2:1 to exactly or about 2.3:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range from about 2:1 to exactly or about 2.2:1.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from just Or a range of about 2:1 to exactly or about 2.1:1.

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or about 2:1.

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is exactly or about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3: 1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8: 1. 4.9:1 or 5:1.

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the number of APCs added in the initial first amplification is exactly or about 1×10 ⁸ , 1.1×10 ⁸ , 1.2 ×10 ⁸ , 1.3×10 ⁸ , 1.4×10 ⁸ , 1.5×10 ⁸ , 1.6×10 ⁸ , 1.7×10 ⁸ , 1.8×10 ⁸ , 1.9×10 ⁸ , 2×10 ⁸ , 2.1×10 ⁸ , 2.2 ×10 ⁸ , 2.3×10 ⁸ , 2.4×10 ⁸ , 2.5×10 ⁸ , 2.6×10 ⁸ , 2.7×10 ⁸ , 2.8×10 ⁸ , 2.9×10 ⁸ , 3×10 ⁸ , 3.1×10 ⁸ , 3.2 ×10 ⁸ , 3.3×10 ⁸ , 3.4×10 ⁸ , or 3.5×10 ⁸ APCs, and wherein the number of APCs added in the rapid second amplification is exactly or approximately 3.5×10 ⁸ , 3.6×10 ⁸ , 3.7×10 ⁸ , 3.8×10 ⁸ , 3.9×10 ⁸ , 4×10 8 , 4.1×10 ⁸ , 4.2×10 ⁸ , 4.3×10 ⁸ , 4.4×10 ⁸ , 4.5×10 ⁸ , 4.6× ^{10 8 ,} 4.7×10 8 ⁸ , 4.8×10 ⁸ , 4.9×10 ⁸ , 5×10 8 , 5.1×10 ⁸ , 5.2×10 ⁸ , 5.3×10 ⁸ , 5.4×10 ⁸ , 5.5×10 ⁸ , 5.6× ^{10 8 ,} 5.7×10 8 ⁸ , 5.8×10 ⁸ , 5.9×10 ⁸ , 6×10 8 , 6.1×10 ⁸ , 6.2×10 ⁸ , 6.3×10 ⁸ , 6.4×10 ⁸ , 6.5×10 ⁸ , 6.6× ^{10 8 ,} 6.7×10 8 ⁸ , 6.8×10 ⁸ , 6.9×10 ⁸ , 7×10 8 , 7.1×10 ⁸ , 7.2×10 ⁸ , 7.3×10 ⁸ , 7.4×10 ⁸ , 7.5×10 ⁸ , 7.6× ^{10 8 ,} 7.7×10 8 ⁸ , 7.8×10 ⁸ , 7.9×10 ⁸ , 8×10 8 , 8.1×10 ⁸ , 8.2×10 ⁸ , 8.3×10 ⁸ , 8.4×10 ⁸ , 8.5×10 ⁸ , 8.6× ^{10 8 ,} 8.7×10 8 ⁸ , 8.8×10 ⁸ , 8.9×10 ⁸ , 9×10 8 , 9.1×10 ⁸ , 9.2×10 ⁸ , 9.3×10 ⁸ , 9.4×10 ⁸ , 9.5×10 ⁸ , 9.6× ^{10 8 ,} 9.7×10 8 ⁸ , 9.8×10 ⁸ , 9.9×10 ⁸ or 1×10 ⁹ APCs.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the number of APCs added in the initial first amplification is selected from exactly or about 1 x ¹⁰⁸ APCs to just or A range of about 3.5×10 ⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of just or about 3.5×10 ⁸ APCs to just or about 1×10 ⁹ APCs.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the number of APCs added in the initial first amplification is selected from exactly or about 1.5 x ¹⁰⁸ APCs to just or A range of about 3×10 ⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of just or about 4×10 ⁸ APCs to just or about 7.5×10 ⁸ APCs.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the number of APCs added in the initial first amplification is selected from exactly or about 2 x ¹⁰⁸ APCs to just or A range of about 2.5 x ¹⁰⁸ APCs, and wherein the number of APCs added in the rapid second amplification is selected from the range of just or about 4.5 x ¹⁰⁸ APCs to just or about 5.5 x ¹⁰⁸ APCs.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein exactly or about 2.5 x ¹⁰ APCs are added to initiate the first amplification, and exactly or about 5 x 10 ⁸ APC lines were added to the rapid second amplification.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the antigen presenting cells are peripheral blood mononuclear cells (PBMCs).

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein a plurality of tumor fragments is distributed into a plurality of separate containers, and in each separate container, the first population of TILs is obtained in step (a), the second population of TILs obtained in step (b), and the third population of TILs obtained in step (c), and the therapeutic TIL population from the plurality of containers in step (c) Combined to produce the pooled TIL population from step (d).

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein a plurality of tumors is evenly distributed into a plurality of separate containers.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein the plurality of separate containers comprises at least two separate containers.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the plurality of separate containers comprises two to twenty separate containers.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the plurality of separate containers comprises two to fifteen separate containers.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the plurality of separate containers comprises two to ten separate containers.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the plurality of separate containers comprises two to five separate containers.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein the plurality of separate containers comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 divided containers.

In other embodiments, the present invention provides a modified method as described in any preceding paragraph as applicable, wherein the initial first amplification of the first population of TILs in step (b) is performed in each vessel, and in the same vessel The rapid second expansion in step (c) is performed on a second population of TILs produced from such a first population of TILs.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein each of the separate containers comprises a first gas permeable surface area.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein multiple tumor fragments are distributed in a single container.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein the single container comprises the first gas permeable surface area.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified such that. In other embodiments, the present invention provides the method described in any of the preceding paragraphs as applicable, modified such that for populations of CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs wherein in step (b) is initiated For each container from which the first amplification was initiated, the second TIL population from such CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population was subjected to the rapid second amplification in step (c) in the same container.

In some embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented by additional antigen-presenting cells (APCs) Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step In (b), the APC is laminated to the first gas permeable surface region at or with an average thickness of from about one cell layer to or to about three cell layers.

In other embodiments, the present invention provides a method described in any preceding paragraph as applicable, modified wherein in step (b), the APCs are present at an average of exactly or about 1.5 cell layers to exactly or about 2.5 cell layers A thickness is laminated to the first air permeable surface area.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein in step (b), the APCs are laminated to the first gas permeable surface region at an average thickness of just or about 2 cell layers superior.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein in step (b), the APC is laminated to the first gas permeable surface region at an average thickness of just or about: 1 , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 cell layers.

In other embodiments, the present invention provides a method described in any preceding paragraph as applicable, modified wherein in step (c), the APCs are present at an average of exactly or about 3 cell layers to exactly or about 10 cell layers A thickness is laminated to the first air permeable surface area.

In other embodiments, the invention provides a method described in any preceding paragraph as applicable, modified wherein in step (c), the APCs are present at an average of exactly or about 4 cell layers to exactly or about 8 cell layers A thickness is laminated to the first air permeable surface area.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (c), the APC is at or about 3, 4, 5, 6, 7, 8, 9 or An average thickness of 10 cell layers was laminated onto the first gas permeable surface area.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein in step (c), the APC is laminated to the first gas permeable surface area at an average thickness of at or about: 4 , 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5 , 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 cell layers.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified wherein in step (b), the first amplification is initiated in a first vessel comprising a first gas permeable surface area is performed, and in step (c), a rapid second amplification is performed in a second vessel comprising a second gas permeable surface area.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the second container is larger than the first container.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). Cell culture medium of the first TIL population, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are at or about one cell layer to just Or an average thickness of about three cell layers is laminated to the first gas permeable surface area.

In other embodiments, the present invention provides a method described in any preceding paragraph as applicable, modified wherein in step (b), the APCs are present at an average of exactly or about 1.5 cell layers to exactly or about 2.5 cell layers A thickness is laminated to the first air permeable surface area.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein in step (b), the APCs are laminated to the first gas permeable surface region at an average thickness of just or about 2 cell layers superior.

In other embodiments, the present invention provides a modification of the method described in any of the preceding paragraphs as applicable, wherein in step (b), the APC is laminated to the first gas permeable surface area at an average thickness of at or about: 1 , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 cell layers.

In other embodiments, the present invention provides a method described in any preceding paragraph as applicable, modified wherein in step (c), the APCs are present at an average of exactly or about 3 cell layers to exactly or about 10 cell layers The thickness is laminated to the second air permeable surface area.

In other embodiments, the invention provides a method described in any preceding paragraph as applicable, modified wherein in step (c), the APCs are present at an average of exactly or about 4 cell layers to exactly or about 8 cell layers The thickness is laminated to the second air permeable surface area.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (c), the APC is at or about 3, 4, 5, 6, 7, 8, 9 or An average thickness of 10 cell layers was laminated onto the second gas permeable surface area.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, modified as appropriate such that in step (c), the APC is at or at about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, An average thickness of 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 cell layers laminated onto the second gas permeable surface area.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified wherein in step (b), the first amplification is initiated in a first vessel comprising a first gas permeable surface area is performed, and in step (c), a rapid second amplification is performed in the first vessel.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). Cell culture medium of the first TIL population, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b), the APCs are at or about one cell layer to just Or an average thickness of about three cell layers is laminated to the first gas permeable surface area.

In other embodiments, the present invention provides a method described in any preceding paragraph as applicable, modified wherein in step (b), the APCs are present at an average of exactly or about 1.5 cell layers to exactly or about 2.5 cell layers A thickness is laminated to the first air permeable surface area.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein in step (b), the APCs are laminated to the first gas permeable surface region at an average thickness of just or about 2 cell layers superior.

In other embodiments, the present invention provides the method described in any preceding paragraph, as applicable above, modified such that in step (b), the APC is at or at about 1, 1.1, 1.2, 1.3, 1.4, 1.5, An average thickness of 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 cell layers laminated onto the first gas permeable surface area.

In other embodiments, the present invention provides a method described in any preceding paragraph as applicable, modified wherein in step (c), the APCs are present at an average of exactly or about 3 cell layers to exactly or about 10 cell layers A thickness is laminated to the first air permeable surface area.

In other embodiments, the invention provides a method described in any preceding paragraph as applicable, modified wherein in step (c), the APCs are present at an average of exactly or about 4 cell layers to exactly or about 8 cell layers A thickness is laminated to the first air permeable surface area.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (c), the APC is at or about 3, 4, 5, 6, 7, 8, 9 or An average thickness of 10 cell layers was laminated onto the first gas permeable surface area.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein in step (c), the APC is laminated to the first gas permeable surface area at an average thickness of at or about: 4 , 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5 , 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 cell layers.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.1 to just or about 1:10.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.1 to just or about 1:9.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.1 to just or about 1:8.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.1 to just or about 1:7.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.1 to just or about 1:6.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.1 to just or about 1:5.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.1 to just or about 1:4.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.1 to just or about 1:3.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.1 to just or about 1:2.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.2 to just or about 1:8.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.3 to just or about 1:7.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.4 to just or about 1:6.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.5 to just or about 1:5.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.6 to just or about 1:4.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.7 to just or about 1:3.5.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.8 to just or about 1:3.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the first expansion is initiated by supplementing with additional antigen presenting cells (APCs). The cell culture medium of the first TIL population is performed, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the average number of layers of APCs stacked in step (b) is the same as in step (c) The ratio of the average number of layers of stacked APCs is selected from the range of just or about 1:1.9 to just or about 1:2.5.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:2.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from exactly or about 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1: 1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:3.8 4. 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1: 6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7, 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:8.8 9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, or 1:10.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.1 to exactly or about 1:10.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.1 to exactly or about 1:9.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.1 to exactly or about 1:8.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.1 to exactly or about 1:7.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.1 to exactly or about 1:6.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.1 to exactly or about 1:5.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.1 to exactly or about 1:4.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.1 to exactly or about 1:3.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.1 to exactly or about 1:2.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.2 to exactly or about 1:8.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.3 to exactly or about 1:7.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.4 to exactly or about 1:6.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.5 to exactly or about 1:5.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.6 to exactly or about 1:4.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable above, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented by additional antigen-presenting cells (APCs) Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.7 to exactly or about 1:3.5.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.8 to exactly or about 1:3.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:1.9 to exactly or about 1:2.5.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from the range of exactly or about 1:2.

In other embodiments, the invention provides the method described in any of the preceding paragraphs, as applicable, modified such that in step (b), the CD39/CD69 doublet of TIL is supplemented with additional antigen-presenting cells (APCs). Negative and/or cell culture medium of CD39 ^LO /CD69 ^LO population to perform primary first expansion, wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step The ratio of the average number of layers of APC laminated in (b) to the average number of layers of APC laminated in step (c) is selected from exactly or about 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1: 1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:3.8 4. 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1: 6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7, 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:8.8 9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, or 1:10.

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is exactly or about 50:1 .

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is exactly or about 25:1 .

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is exactly or about 20:1 .

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is exactly or about 10:1 .

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein the second population of TILs is at least exactly or about 50 times greater in number than the first population of TILs.

In other embodiments, the invention provides a method described in any preceding paragraph as applicable, modified wherein the second population of TILs is at least exactly or about 1, 2, 3, 4, 5 greater in number than the first population of TILs , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times.

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified wherein exactly or about 2 days or exactly or about 3 days after the start of the second period of time in step (c), the cells Medium was supplemented with additional IL-2.

In other embodiments, the invention provides a modified version of the method described in any preceding paragraph as applicable, further comprising the step of cryopreserving the collected TIL population in step (d) using a cryopreservation process.

In other embodiments, the present invention provides a modification of the method described in any of the preceding paragraphs as applicable above, comprising carrying out after step (d) the transfer of the collected TIL population from step (d) to an optionally containing HypoThermosol Additional step (e) of the infusion bag.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, comprising the step of cryopreserving the infusion bag comprising the collected TIL population in step (e) using a cryopreservation process.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the cryopreservation process is performed using a 1:1 ratio of the collected TIL population to cryopreservation medium.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the antigen presenting cells are peripheral blood mononuclear cells (PBMCs).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the PBMCs are irradiated and allogeneic.

In other embodiments, the invention provides a modification of the method described in any preceding paragraph as applicable, wherein the total number of APCs added to the cell culture in step (b) is 2.5 x ¹⁰⁸ .

In other embodiments, the invention provides a modification of the method described in any preceding paragraph as applicable, wherein the total number of APCs added to the cell culture in step (c) is 5 x ¹⁰⁸ .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the APCs are PBMCs.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the PBMCs are irradiated and allogeneic.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the antigen presenting cell is an artificial antigen presenting cell.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the collection in step (d) is performed using a membrane-based cell processing system.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the harvesting in step (d) is performed using the LOVO cell processing system.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein the plurality of fragments comprises exactly or about 5 to exactly or about 60 fragments per container in step (b).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein the plurality of fragments comprises exactly or about 10 to exactly or about 60 fragments per container in step (b).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein the plurality of fragments comprises exactly or about 15 to exactly or about 60 fragments per container in step (b).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein the plurality of fragments comprises exactly or about 20 to exactly or about 60 fragments per container in step (b).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein the plurality of fragments comprises exactly or about 25 to exactly or about 60 fragments per container in step (b).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein the plurality of fragments comprises exactly or about 30 to exactly or about 60 fragments per container in step (b).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein the plurality of fragments comprises exactly or about 35 to exactly or about 60 fragments per container in step (b).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein the plurality of fragments comprises exactly or about 40 to exactly or about 60 fragments per container in step (b).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein the plurality of fragments comprises exactly or about 45 to exactly or about 60 fragments per container in step (b).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein the plurality of fragments comprises exactly or about 50 to exactly or about 60 fragments per container in step (b).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein a plurality of fragments is comprised in step (b) of just or about 2, 3, 4, 5, 6, 7 per container , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59 or 60 clips.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein each fragment has a volume of exactly or about 27 mm ³ .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein each segment has a volume of exactly or about 20 mm ³ to just or about 50 mm ³ .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein each fragment has a volume of exactly or about 21 mm ³ to exactly or about 30 mm ³ .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein each fragment has a volume of exactly or about 22 mm ³ to exactly or about 29.5 mm ³ .

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein each segment has a volume of exactly or about 23 mm ³ to exactly or about 29 mm ³ .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein each fragment has a volume of exactly or about 24 mm ³ to exactly or about 28.5 mm ³ .

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein each segment has a volume of exactly or about 25 mm ³ to exactly or about 28 mm ³ .

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein each fragment has a volume of exactly or about 26.5 mm ³ to exactly or about 27.5 mm ³ .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein each fragment has a volume of exactly or about: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mm ³ .

In other embodiments, the present invention provides a method as described in any preceding paragraph, as applicable, modified such that a plurality of fragments comprises or comprises from about 30 to or to about 60 fragments, wherein the total volume is or is about 1300 mm ³ to or to about 1500 mm ³ .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the plurality of fragments comprises exactly or about 50 fragments, wherein the total volume is exactly or about 1350 mm ³ .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein the plurality of fragments comprises exactly or about 50 fragments, wherein the total mass is exactly at or about 1 gram to exactly or about 1.5 grams .

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the cell culture medium is provided in a container that is a G container or a Xuri cell bag.

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the concentration of IL-2 in the cell culture medium is from about 10,000 IU/mL to about 5,000 IU/mL.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the concentration of IL-2 in the cell culture medium is about 6,000 IU/mL.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the cryopreservation medium comprises dimethylsulfoxide (DMSO).

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the cryopreservation medium comprises 7% to 10% DMSO.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the first period of time in step (b) is exactly or about 1 day, 2 days, 3 days, 4 days, 5-day, 6-day or 7-day period.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the second period of time in step (c) is at or about 1 day, 2 days, 3 days, 4 days, 5-day, 6-day, 7-day, 8-day, 9-day, 10-day or 11-day period.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first period of time in step (b) and the second period of time in step (c) are each at exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first period of time in step (b) and the second period of time in step (c) are each at exactly or about 5-day, 6-day or 7-day period.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first period of time in step (b) and the second period of time in step (c) are each at exactly or about within a 7-day period.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed for a total of exactly or about 14 days to exactly or about 18 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed for a total of exactly or about 15 days to exactly or about 18 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed for a total of exactly or about 16 days to exactly or about 18 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed for a total of exactly or about 17 days to exactly or about 18 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed for a total of exactly or about 14 days to exactly or about 17 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed for a total of exactly or about 15 days to exactly or about 17 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein steps (a) to (d) are performed for a total of exactly or about 16 days to exactly or about 17 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed for a total of exactly or about 14 days to exactly or about 16 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed for a total of exactly or about 15 days to exactly or about 16 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed over a total of exactly or about 14 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed over a total of exactly or about 15 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed over a total of exactly or about 16 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein steps (a) to (d) are performed over a total of exactly or about 17 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein steps (a) to (d) are performed over a total of exactly or about 18 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed in a total of exactly or about 14 days or less.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed in a total of exactly or about 15 days or less.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed in a total of exactly or about 16 days or less.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein steps (a) to (d) are performed in a total of exactly or about 18 days or less.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein the population of therapeutic TILs collected in step (d) comprises a therapeutically effective dose of TILs sufficient for TILs.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein the number of TILs sufficient for a therapeutically effective dose is from exactly or about 2.3 x ¹⁰¹⁰ to exactly or about 13.7 x ¹⁰¹⁰ indivual.

In other embodiments, the invention provides a method described in any preceding paragraph as applicable, modified wherein the third TIL population in step (c) provides increased efficacy, increased interferon-γ production and/or increased of polyploidy.

In other embodiments, the invention provides a method described in any preceding paragraph as applicable, modified wherein the third population of TILs in step (c) provides At least one-fold to five-fold or more interferon-γ production.

In other embodiments, the invention provides a method described in any preceding paragraph as applicable, modified wherein the third population of TILs in step (c) provides At least one-fold to five-fold or more interferon-γ production.

In other embodiments, the invention provides a method described in any preceding paragraph as applicable, modified wherein the third population of TILs in step (c) provides At least one-fold to five-fold or more interferon-γ production.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein relative to the effector T cells and/or central memory T cells obtained from the second cell population in step (b), obtained from Step (c) effector T cells and/or central memory T cells of the third TIL population exhibit increased CD8 and CD28 expression.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, as modified, wherein each container recited in the method is a closed container.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein each receptacle recited in the method is a G receptacle.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein each vessel recited in the method is GREX-10.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein each vessel recited in the method is GREX-100.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein each vessel recited in the method is GREX-500.

In other embodiments, the present invention provides a therapeutic tumor infiltrating lymphocyte (TIL) population prepared by the method described in any preceding paragraph as applicable.

In other embodiments, the present invention provides a therapeutic tumor infiltrating lymphocyte (TIL) population prepared from tumor tissue of a patient, wherein compared to by wherein the first expansion of TIL is in the absence of any added antigen presentation Cells (APC) or OKT3 produced by the process, the therapeutic TIL population provides increased potency, increased interferon-gamma production and/or increased polyclonality.

In other embodiments, the present invention provides a therapeutic tumor infiltrating lymphocyte (TIL) population prepared from tumor tissue of a patient, wherein compared to by wherein the first expansion of TIL is in the absence of any added antigen presentation In the case of TILs produced by the process performed in the case of cells (APCs), the therapeutic TIL population provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality.

In other embodiments, the present invention provides a therapeutic tumor infiltrating lymphocyte (TIL) population prepared from tumor tissue of a patient, wherein compared to that by wherein the first expansion of TIL is in the absence of any added OKT3 Where TILs are produced by the process, the therapeutic TIL population provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from tumor tissue of a patient, wherein compared to that by wherein the first expansion of TIL is in the absence of added antigen-presenting cells (APC) and TILs prepared by the process performed without added OKT3, the therapeutic TIL population provided increased efficacy, increased interferon-γ production, and/or increased polyclonality.

In other embodiments, the present invention provides a population of therapeutic tumor infiltrating lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the population of therapeutic TILs provides Increased efficacy, increased interferon-gamma production and/or increased polyclonality.

In other embodiments, the invention provides a population of therapeutic tumor infiltrating lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the population of therapeutic TILs provides Increased efficacy, increased interferon-gamma production and/or increased polyclonality.

In other embodiments, the invention provides a population of therapeutic tumor infiltrating lymphocytes (TILs) prepared from tumor tissue of a patient, wherein the population of therapeutic TILs provides Increased efficacy, increased interferon-gamma production and/or increased polyclonality.

In other embodiments, the invention provides a population of therapeutic TILs as described in any preceding paragraph as applicable, which population of therapeutic TILs provides increased interferon-gamma production.

In other embodiments, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs, as applicable, that provides increased polyclonality.

In other embodiments, the present invention provides a therapeutic TIL population as described in any of the preceding paragraphs, as applicable, that provides increased efficacy.

In other embodiments, the invention provides a modified version of the therapeutic TIL population described in any preceding paragraph as applicable, wherein the therapeutic TIL population is capable of producing at least more Twice as much interferon-gamma. In other embodiments, the invention provides a modified therapeutic TIL population as described in any preceding paragraph as applicable, wherein the therapeutic TIL population is capable of producing at least more TILs than TILs produced by a process longer than 17 days Twice as much interferon-gamma. In other embodiments, the invention provides a modified version of the therapeutic TIL population described in any preceding paragraph as applicable, wherein the therapeutic TIL population is capable of producing at least more Twice as much interferon-gamma. In some embodiments, due to the amplification procedure described herein, for example as described in Steps A to F above or according to Steps A to F above (also as for example in FIG. 8 (especially for example in FIG. 8A and/or or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ), TILs appear to be able to produce at least one-fold higher interferon-γ.

In other embodiments, the invention provides a modified version of the therapeutic TIL population described in any preceding paragraph as applicable, wherein the therapeutic TIL population is capable of producing at least more Twice as much interferon-gamma. In other embodiments, the invention provides a modified therapeutic TIL population as described in any preceding paragraph as applicable, wherein the therapeutic TIL population is capable of producing at least more TILs than TILs produced by a process longer than 17 days Twice as much interferon-gamma. In other embodiments, the invention provides a modified version of the therapeutic TIL population described in any preceding paragraph as applicable, wherein the therapeutic TIL population is capable of producing at least more Twice as much interferon-gamma. In some embodiments, due to the amplification procedure described herein, for example as described in Steps A to F above or according to Steps A to F above (also as for example in FIG. 8 (especially for example in FIG. 8A and/or or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ), TILs appear to be able to produce at least two times higher interferon-γ.

In other embodiments, the invention provides a modified version of the therapeutic TIL population described in any preceding paragraph as applicable, wherein the therapeutic TIL population is capable of producing at least more than three times the amount of interferon-gamma. In other embodiments, the invention provides a modified therapeutic TIL population as described in any preceding paragraph as applicable, wherein the therapeutic TIL population is capable of producing at least more TILs than TILs produced by a process longer than 17 days than three times the amount of interferon-gamma. In other embodiments, the invention provides a modified version of the therapeutic TIL population described in any preceding paragraph as applicable, wherein the therapeutic TIL population is capable of producing at least more than three times the amount of interferon-gamma. In some embodiments, due to the amplification procedure described herein, for example as described in Steps A to F above or according to Steps A to F above (also as for example in FIG. 8 (especially for example in FIG. 8A and/or or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ), TILs appear to be able to produce at least three times higher interferon-γ.

In other embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population compared to a method wherein the first expansion of TIL is performed without any added antigen-presenting cells (APCs). The therapeutic tumor infiltrating lymphocyte population is capable of producing at least one-fold more interferon-gamma from TILs produced by the process. In some embodiments, due to the amplification procedure described herein, for example as described in Steps A to F above or according to Steps A to F above (also as for example in FIG. 8 (especially for example in FIG. 8A and/or or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ), TILs appear to be able to produce at least one-fold higher interferon-γ.

In other embodiments, the present invention provides a therapeutic tumor infiltrating lymphocyte (TIL) population compared to TIL prepared by a process wherein the first expansion of TIL is performed without any added OKT3 , the therapeutic tumor infiltrating lymphocyte population produces at least twice as much interferon-gamma. In some embodiments, due to the amplification procedure described herein, for example as described in Steps A to F above or according to Steps A to F above (also as for example in FIG. 8 (especially for example in FIG. 8A and/or or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ), TILs appear to be able to produce at least one-fold higher interferon-γ.

In other embodiments, the invention provides a population of therapeutic TILs that produces At least twice as much interferon-gamma. In some embodiments, due to the amplification procedure described herein, for example as described in Steps A to F above or according to Steps A to F above (also as for example in FIG. 8 (especially for example in FIG. 8A and/or or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ), TILs appear to be able to produce at least two times higher interferon-γ.

In other embodiments, the present invention provides a population of therapeutic TILs that produces At least twice as much interferon-gamma. In some embodiments, due to the amplification procedure described herein, for example as described in Steps A to F above or according to Steps A to F above (also as for example in FIG. 8 (especially for example in FIG. 8A and/or or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ), TILs appear to be able to produce at least two times higher interferon-γ.

In other embodiments, the invention provides a population of therapeutic TILs that produces At least three times more interferon-gamma. In some embodiments, due to the amplification procedure described herein, for example as described in Steps A to F above or according to Steps A to F above (also as for example in FIG. 8 (especially for example in FIG. 8A and/or or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ), TILs appear to be able to produce at least one-fold higher interferon-γ.

In other embodiments, the present invention provides a population of therapeutic TILs that produces At least three times more interferon-gamma. In some embodiments, due to the amplification procedure described herein, for example as described in Steps A to F above or according to Steps A to F above (also as for example in FIG. 8 (especially for example in FIG. 8A and/or or FIG. 8B and/or FIG. 8C and/or FIG. 8D and/or FIG. 8E and/or FIG. 8F and/or FIG. 8G ), TILs appear to be able to produce at least three times higher interferon-γ.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a punch biopsy), a core needle biopsy, a core needle biopsy, or a fine needle Aspirate.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein the tumor fragment is a core needle biopsy.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein the tumor fragment is a fine needle aspirate.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the tumor fragment is a small biopsy (including, for example, a punch biopsy).

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the tumor fragment is a core needle biopsy.

In other embodiments, the invention provides a method as described in any preceding paragraph, as applicable, modified such that (i) the method comprises one or more small biopsies from tumor tissue from an individual (including, for example, punch biopsy), core needle biopsy, core needle biopsy, or fine needle aspirate to obtain the first TIL population, (ii) the method comprises, prior to performing the initial first amplification step, performing the step of culturing the first TIL population in IL-2 cell culture medium for a period of about 3 days, (iii) the method comprising performing the initial first expansion for a period of about 8 days, and (iv) the method comprising performing a rapid The second time period of expansion is about 11 days. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In other embodiments, the invention provides a method as described in any preceding paragraph, as applicable, modified such that (i) the method comprises one or more small biopsies from tumor tissue from an individual (including, for example, punch biopsy), core needle biopsy, core needle biopsy, or fine needle aspirate to obtain the first TIL population, (ii) the method comprises, prior to performing the initial first amplification step, performing the step of culturing the first TIL population in IL-2 cell culture medium for a period of about 3 days, (iii) the method comprising performing the initial first expansion for a period of about 8 days, and (iv) the method comprising performing the initial expansion by A culture of the second TIL population is grown for about 5 days, the culture is divided into up to 5 subcultures and the subcultures are grown for about 6 days to perform a rapid second expansion. In some of the foregoing embodiments, up to 5 subcultures are each grown in a vessel the same size or larger than the vessel in which the second population of TILs was initially cultured in the rapid second expansion. In some of the foregoing embodiments, the culture of the second population of TILs is divided equally among at most 5 subcultures. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In other embodiments, the invention provides a method as described in any preceding paragraph, as applicable, modified such that the first population of TILs is obtained from 1 to about 20 small biopsies of tumor tissue from an individual (including, for example, punch biopsy), core-needle biopsy, core-needle biopsy, or fine-needle aspirate.

In other embodiments, the invention provides a method as described in any preceding paragraph, as applicable, modified such that the first population of TILs is obtained from 1 to about 10 small biopsies of tumor tissue from an individual (including, for example, punch biopsy), core-needle biopsy, core-needle biopsy, or fine-needle aspirate.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is derived from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 small biopsies (including, for example, punch biopsies), core biopsy, core needle aspiration Obtained by biopsy or fine needle aspirate.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is derived from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 small biopsies (including, for example, punch biopsies), core needle biopsies, core needle biopsies, or fine needle aspirations.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is obtained from 1 to about 20 core biopsies of tumor tissue from an individual.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is obtained from 1 to about 10 core biopsies of tumor tissue from an individual.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is derived from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 core needle biopsies were obtained.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is derived from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 core-needle biopsies were obtained.

In other embodiments, the present invention provides the method described in any preceding paragraph, as applicable, modified such that the first population of TILs is obtained from 1 to about 20 fine needle aspirates of tumor tissue from an individual.

In other embodiments, the present invention provides the method described in any preceding paragraph, as applicable, modified such that the first population of TILs is obtained from 1 to about 10 fine needle aspirates of tumor tissue from an individual.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is derived from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 FNAs were obtained.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is derived from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 FNAs were obtained.

In other embodiments, the invention provides a method as described in any preceding paragraph, as applicable, modified such that the first population of TILs is obtained from 1 to about 20 core needle biopsies of tumor tissue from an individual .

In other embodiments, the invention provides a method as described in any preceding paragraph, as applicable, modified such that the first population of TILs is obtained from 1 to about 10 core needle biopsies of tumor tissue from an individual .

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is derived from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 core-needle biopsies were obtained.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is derived from 1, 2, 3, 4, 5, 6, Obtained from 7, 8, 9 or 10 core-needle biopsies.

In other embodiments, the invention provides a method as described in any preceding paragraph, as applicable, modified such that the first population of TILs is obtained from 1 to about 20 small biopsies of tumor tissue from an individual (including, for example, perforated biopsy).

In other embodiments, the invention provides a method as described in any preceding paragraph, as applicable, modified such that the first population of TILs is obtained from 1 to about 10 small biopsies of tumor tissue from an individual (including, for example, perforated biopsy).

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is derived from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 small biopsies (including for example punched biopsies) are obtained.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the first population of TILs is derived from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 small biopsies (including eg punch biopsies) are obtained.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified such that (i) the method comprises obtaining a first TIL from 1 to about 10 core needle biopsies of tumor tissue from an individual population; (ii) the method comprising the step of culturing the first TIL population in a cell culture medium comprising IL-2 prior to the step of isolating the CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population from the first TIL population TIL population for a period of about 3 days; (iii) the method comprises culturing CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs in a medium comprising IL-2, OKT-3 and antigen presenting cells (APCs) A period of about 8 days for the population to initiate the first expansion step to obtain a second population of TILs; and (iv) the method comprises culturing the second TILs in a medium comprising IL-2, OKT-3, and APCs The population was approximately 11 days old for a rapid second expansion. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified such that (i) the method comprises obtaining a first TIL from 1 to about 10 core needle biopsies of tumor tissue from an individual population; (ii) the method comprising the step of culturing the first TIL population in a cell culture medium comprising IL-2 prior to the step of isolating the CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population from the first TIL population TIL population for a period of about 3 days; (iii) the method comprises culturing CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TILs in a medium comprising IL-2, OKT-3 and antigen presenting cells (APCs) A period of about 8 days for the population to undergo an initial first expansion step to obtain a second TIL population; and (iv) the method comprises performing a rapid second expansion by: The culture of the second TIL population was grown in medium containing IL-2 for about 5 days, the culture was divided into up to 5 subcultures and each of these subcultures was cultured in medium comprising IL-2 for about 5 days. 6 days. In some of the foregoing embodiments, up to 5 subcultures are each grown in a vessel the same size or larger than the vessel in which the second population of TILs was initially cultured in the rapid second expansion. In some of the foregoing embodiments, the culture of the second population of TILs is divided equally among at most 5 subcultures. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified such that (i) the method comprises obtaining a first TIL from 1 to about 10 core needle biopsies of tumor tissue from an individual population; (ii) the method comprises the following steps prior to the step of isolating the CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population from the first TIL population: In a G-REX-100M culture flask, There is a period of about 3 days in which the first TIL population is cultured in cell culture medium containing 0.5 L of CM1 medium containing 6000 IU IL-2/mL; (iii) the method comprises a CD39/CD69 double negative and/or CD39 ^LO /CD69 0.5 L CM1 medium containing 6000 IU/mL IL-2, 30 ng/mL OKT-3 and about 10 ⁸ feeder cells was added to the ^LO TIL population and cultured for about 8 days to initiate the first expansion to generating a second TIL population, and (iv) the method comprising performing a rapid second amplification by (a) combining the second TIL population with 3000 IU/mL IL-2, 30 ng/mL OKT-3, and 5×10 ⁹ feeder cells were transferred together to a G-REX 500MCS culture flask containing 5 L CM2 medium and cultured for about 5 days, (b) by transferring 10 ⁹ TILs together with 3000 IU/mL IL-2 to Split the culture into up to 5 subcultures in each of up to 5 G-REX 500MCS flasks containing 5 L of AIM-V medium and grow the subcultures for about 6 days. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In some embodiments, the invention provides a method as described in any preceding paragraph as applicable, such that (a) after the step of obtaining a tumor sample from an individual, (i) culturing the tumor sample in a cell culture medium containing IL-2 The subject TIL or first TIL population in, to produce TIL released from tumor fragments or samples, (ii) isolating at least a plurality of TILs released from tumor fragments or samples from tumor fragments or samples, to produce tumor fragments or samples , a mixture of TIL remaining in the tumor fragment or sample and any TIL released from the tumor fragment or sample and remaining with it after isolation, (iii) combining the tumor fragment or sample, the remaining TIL in the tumor fragment or sample and from Digestion of mixtures of any TILs released from tumor fragments or samples and remaining with them after isolation to produce digests of such mixtures; and (iv) isolation of CD39/CD69 double negative and/or CD39 from digests of such mixtures ^LO /CD69 ^LO TIL populations; and (b) initial first expansion by culturing CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL populations. In some embodiments, at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 95%, 99% or higher percentage of TIL released from tumor fragments or samples is isolated from the tumor fragments or samples to produce a mixture.

In some embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified such that the culturing step prior to initiating the first amplification is performed for a period of about 1 day to about 3 days.

In some embodiments, the invention provides a method as described in any preceding paragraph as applicable above, modified such that the culturing step performed prior to initiating the first amplification is performed for about 1, 2, 3, 4, 5 , 6 or 7 days.

In some embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the culturing step prior to the PD-1 preselection step is performed for a period of about 1 day to about 3 days.

In some embodiments, the invention provides a method as described in any preceding paragraph, as applicable, modified such that the culturing step prior to the PD-1 preselection step is performed by about 1, 2, 3, 4, 5, 6 or 7 day period.

In some embodiments, the invention provides a method as described in any preceding paragraph as applicable, such that the culturing step preceding the CD39/CD69 preselection step is performed for a period of about 1 day to about 3 days.

In some embodiments, the invention provides a method as described in any of the preceding paragraphs where applicable, such that the culturing step prior to the CD39/CD69 preselection step is performed within about 1, 2, 3, 4, 5, 6, or 7 days time period.

In other embodiments, the present invention provides a method for expanding T cells, comprising: (a) priming a first T cell population obtained from a donor by culturing the first T cell population; Expansion to achieve growth and initiate activation of the first T cell population; (b) after the activation of the first T cell population initiated in step (a) has begun to decline, performing the first T cell population by culturing the first T cell population rapid second expansion of the cell population to effect growth and enhance activation of the first T cell population to obtain a second T cell population; and (c) collecting the second T cell population. In other embodiments, the step of rapid second expansion is divided into multiple steps to achieve vertical scale-up of culture by: (a) by A rapid second expansion is performed by culturing the first T cell population in small culture for a period of about 3 to 4 days; and then (b) effectuating the transfer of the first T cell population from the small culture to a larger population than the first The first T cell population from the small scale culture is cultured in the larger scale culture for about 4 to 7 days in a second, larger vessel (e.g., G-REX-500MCS vessel) period of time. In other embodiments, the rapid expansion step is divided into multiple steps to achieve culture scale-up by: (a) by using a first small scale in a first vessel (eg, a G-REX-100MCS vessel) The culture grows the first T cell population for a period of about 3 to 4 days to perform a rapid second expansion; and then (b) achieves transfer and distribution of the first T cell population from the first small scale culture to at least 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers equal in size to the first container, wherein In each second vessel, the portion of the first T cell population from the first mini-culture transferred to such second vessel is cultured in the second mini-culture for a period of about 4 to 7 days. In other embodiments, the rapid expansion step is divided into multiple steps to achieve culture scale-up and scale-up by: (a) by A rapid second expansion is performed by culturing the first T cell population in small culture for a period of about 3 to 4 days; and then (b) effecting the transfer and distribution of the first T cell population from the small culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers ( For example, in G-REX-500MCS containers), wherein in each second container, the portion of the first T cell population from a small-scale culture transferred to such second container is cultured in a larger-scale culture for about 4 to 7 time of day. In other embodiments, the rapid expansion step is divided into multiple steps to achieve culture scale-up and scale-up by: (a) by A rapid second expansion is performed by culturing the first T cell population in small culture over a period of about 4 days; and then (b) effecting transfer and distribution of the first T cell population from the small culture to at least 2, Among 3 or 4 second containers (eg, G-REX-500MCS containers) larger in size than the first container, wherein in each second container, the first sample from the small-scale culture transferred to such second container A portion of the T cell population was cultured in larger cultures for a period of about 5 days.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the rapid second expansion step is divided into a plurality of steps to achieve vertical scale-up of the culture by: (a) Rapid second expansion by culturing the first T cell population in small scale culture in the first vessel (e.g. G-REX 100MCS vessel) for a period of about 2 to 4 days; The first T cell population is transferred to a second container larger than the first container (e.g., G-REX 500MCS container), and the first T cells from the small-scale culture are cultured in a larger-scale culture in the second container Groups have a period of about 5 to 7 days.

In other embodiments, the invention provides a modification of the method described in any preceding paragraph as applicable, wherein the rapid expansion step is divided into a plurality of steps to achieve culture scale-out by: (a) by culturing the first T cell population in the first small-scale culture in the first container (e.g., the G-REX 100MCS container) for a period of about 2 to 4 days for a rapid second expansion; The first T cell population of scale culture is transferred and distributed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 in second containers of equal size to the first container, wherein in each second container the portion of the first T cell population from the first small-scale culture transferred to such second container is in the second small-scale culture Cultivate for a period of about 5 to 7 days.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the rapid expansion step is divided into a plurality of steps to achieve culture scale-up and scale-up by: (a ) performing a rapid second expansion by culturing the first T cell population in small scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 2 to 4 days; The first T cell population of scale culture is transferred and distributed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 Among the second containers (such as G-REX 500MCS containers) larger in size than the first container, wherein in each second container, the portion of the first T cell population from small-scale culture transferred to such second container is in A period of about 5 to 7 days is cultivated in larger scale cultures.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the rapid expansion step is divided into a plurality of steps to achieve culture scale-up and scale-up by: (a ) performing a rapid second expansion by culturing the first T cell population in small scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 3 to 4 days; The large-scale cultured first T cell population is transferred and distributed into 2, 3 or 4 second containers (e.g. G-REX 500MCS containers) larger in size than the first container, wherein in each second container, transfer to such The fraction of the first T cell population from the small scale culture in the second vessel is cultured in the larger scale culture for a period of about 5 to 6 days.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the rapid expansion step is divided into a plurality of steps to achieve culture scale-up and scale-up by: (a ) performing a rapid second expansion by culturing the first T cell population in small scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 3 to 4 days; The large-scale cultured first T cell population is transferred and distributed into 2, 3 or 4 second containers (e.g. G-REX 500MCS containers) larger in size than the first container, wherein in each second container, transfer to such The fraction of the first T cell population from the small scale culture in the second vessel is cultured in the larger scale culture for a period of about 5 days.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the rapid expansion step is divided into a plurality of steps to achieve culture scale-up and scale-up by: (a ) performing a rapid second expansion by culturing the first T cell population in small scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 3 to 4 days; The large-scale cultured first T cell population is transferred and distributed into 2, 3 or 4 second containers (e.g. G-REX 500MCS containers) larger in size than the first container, wherein in each second container, the The portion of the first T cell population from the small scale culture in the second vessel is cultured in the larger scale culture for a period of about 6 days.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the rapid expansion step is divided into a plurality of steps to achieve culture scale-up and scale-up by: (a ) performing a rapid second expansion by culturing the first T cell population in small scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 3 to 4 days; The large-scale cultured first T cell population is transferred and distributed into 2, 3 or 4 second containers (e.g. G-REX 500MCS containers) larger in size than the first container, wherein in each second container, the The portion of the first T cell population from the small scale culture in the second vessel is cultured in the larger scale culture for a period of about 7 days.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that the initial first amplification step is performed during a period of at most 7 days.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified such that the rapid second amplification step is performed during a period of up to 8 days.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the rapid second amplification of step (b) is performed over a period of up to 9 days.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the rapid second amplification of step (b) is performed over a period of up to 10 days.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the rapid second amplification of step (b) is performed over a period of up to 11 days.

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the initial first amplification in step (a) is performed over a period of 7 days, and step (b) The rapid second amplification was performed over a period of up to 9 days.

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the initial first amplification in step (a) is performed over a period of 7 days, and step (b) The rapid second amplification was performed over a period of up to 10 days.

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the initial first amplification in step (a) is performed over a period of 7 days or 8 days, and step The rapid second amplification of (b) is performed over a period of up to 9 days.

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the initial first amplification in step (a) is performed over a period of 7 days or 8 days, and step The rapid second amplification of (b) is performed over a period of up to 10 days.

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the initial first amplification in step (a) is performed over a period of 8 days, and step (b) The rapid second amplification was performed over a period of up to 9 days.

In other embodiments, the invention provides a modified method described in any preceding paragraph as applicable, wherein the initial first amplification in step (a) is performed over a period of 8 days, and step (b) The rapid second amplification was performed over a period of up to 8 days.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable, wherein in step (a), the first population of T cells is cultured in a first culture medium comprising OKT-3 and IL-2 cultivated in.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein the first culture medium comprises a 4-1BB agonist, OKT-3 and IL-2.

In other embodiments, the present invention provides a modified method as described in any preceding paragraph as applicable, wherein the first culture medium comprises OKT-3, IL-2 and antigen presenting cells (APCs).

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable, wherein the first culture medium comprises a 4-1BB agonist, OKT-3, IL-2, and antigen presenting cells (APCs).

In other embodiments, the present invention provides a modification of the method described in any of the preceding paragraphs as applicable, wherein in step (b), the first population of T cells is comprised of OKT-3, IL-2, and antigen-presenting cells (APC) in the second culture medium.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable, wherein the second culture medium comprises a 4-1BB agonist, OKT-3, IL-2, and antigen presenting cells (APCs).

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable, wherein in step (a), the first T cell population is in a first culture medium in a vessel comprising a first gas permeable surface Cultured in medium, wherein the first medium comprises OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the donor of the first T cell population, and the first APC The population is layered onto the first gas permeable surface, wherein in step (b), the first T cell population is cultured in a container in a second culture medium comprising OKT-3, IL-2 and a second APC population , wherein the second APC population is exogenous to the donor of the first T cell population, and the second APC population is laminated to the first gas permeable surface, and wherein the second APC population is larger than the first APC population.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable, wherein in step (a), the first T cell population is in a first culture medium in a vessel comprising a first gas permeable surface culture medium, wherein the first medium comprises a 4-1BB agonist, OKT-3, IL-2, and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the first T cell population donor and the first APC population is layered onto the first gas permeable surface, wherein in step (b), the first T cell population is cultured in a container in a second culture medium, wherein the second culture medium comprises OKT-3, IL- 2 and the second population of APCs, wherein the second population of APCs is exogenous to the donor of the first T cell population, and the second population of APCs is laminated onto the first gas permeable surface, and wherein the second population of APCs is larger than the first population of APCs The group is large.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable, wherein in step (a), the first T cell population is in a first culture medium in a vessel comprising a first gas permeable surface Cultured in medium, wherein the first medium comprises OKT-3, IL-2 and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the donor of the first T cell population, and the first APC The population is layered onto the first gas permeable surface, wherein in step (b), the first T cell population is cultured in a vessel in a second culture medium comprising 4-1BB agonist, OKT-3, IL -2 and the second population of APCs, wherein the second population of APCs is exogenous to the donor of the first T cell population, and the second population of APCs is laminated onto the first gas permeable surface, and wherein the second population of APCs is larger than the first population of APCs The APC group is large.

In other embodiments, the invention provides a modified method as described in any preceding paragraph as applicable, wherein in step (a), the first T cell population is in a first culture medium in a vessel comprising a first gas permeable surface culture medium, wherein the first medium comprises a 4-1BB agonist, OKT-3, IL-2, and a first antigen-presenting cell (APC) population, wherein the first APC population is exogenous to the first T cell population donor and the first APC population is laminated onto the first gas permeable surface, wherein in step (b), the first T cell population is cultured in a container in a second culture medium, wherein the second culture medium comprises a 4-1BB agonist , OKT-3, IL-2, and a second APC population, wherein the second APC population is exogenous to the donor of the first T cell population, and the second APC population is laminated onto the first gas permeable surface, and wherein the second APC population is exogenous to the donor of the first T cell population, and wherein the second APC population is The second APC population is larger than the first APC population.

In other embodiments, the present invention provides a modified method described in any preceding paragraph as applicable, wherein the ratio of the number of APCs in the second APC population to the number of APCs in the first APC population is about 2:1 .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the number of APCs in the first APC population is about 2.5×10 ⁸ , and the number of APCs in the second APC population is about 2.5×10 8 . is about 5×10 ⁸ .

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein in step (a), the first APC population is laminated to the first gas permeable surface at an average thickness of 2 APC layers .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the second population of APCs has an average thickness selected from the range of 4 to 8 APC layers Laminated onto the first breathable surface.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the average number of APC layers laminated to the first gas permeable surface in step (b) is the same as in step (a) The ratio of the average number of APC layers laminated onto the first gas permeable surface was 2:1.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (a), the first population of APCs is selected from at or about 1.0×10 ⁶ APCs/cm ² to The first gas permeable surface is seeded at a density in the range of or about 4.5 x ¹⁰⁶ APCs/ ^cm2 .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein in step (a), the first population of APCs is selected from exactly or about 1.5 x ¹⁰⁶ APCs/ ^cm2 A density in the range of just or about 3.5 x ¹⁰⁶ APCs/ ^cm2 is seeded on the first gas permeable surface.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (a), the first population of APCs is selected from at or about 2.0×10 ⁶ APCs/cm ² to The first gas permeable surface is seeded at a density in the range of or about 3.0 x ¹⁰⁶ APCs/ ^cm2 .

In other embodiments, the present invention provides a method described in any preceding paragraph as applicable, modified wherein in step (a), the first APC population is present at a density of exactly or about 2.0 x ¹⁰⁶ APCs/ ^cm2 Inoculated on the first gas permeable surface.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein in step (b), the second population of APCs is selected from exactly or about 2.5 x ¹⁰⁶ APCs/ ^cm2 A density in the range of just or about 7.5 x ¹⁰⁶ APCs/ ^cm2 is seeded on the first gas permeable surface.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein in step (b), the second population of APCs is selected from exactly or about 3.5 x ¹⁰⁶ APCs/ ^cm2 A density in the range of just or about 6.0 x ¹⁰⁶ APCs/ ^cm2 is seeded on the first gas permeable surface.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified wherein in step (b), the second population of APCs is selected from exactly or about 4.0 x ¹⁰⁶ APCs/ ^cm2 A density in the range of just or about 5.5 x ¹⁰⁶ APCs/ ^cm2 is seeded on the first gas permeable surface.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein in step (b), the second APC population is present at a density of exactly or about 4.0 x ¹⁰⁶ APCs/ ^cm2 Inoculated on the first gas permeable surface.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified wherein in step (a), the first population of APCs is selected from exactly or about 1.0 x ¹⁰⁶ APCs/ ^cm2 The first gas permeable surface is seeded on the first gas permeable surface at a density in the range of exactly or about 4.5 x ¹⁰ APCs/cm ² , and in step (b), the second APC population is selected from the group consisting of exactly or about 2.5 x ¹⁰ APCs APC/cm ² to just or about 7.5×10 ⁶ APC/cm ² are seeded on the first gas permeable surface.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein in step (a), the first population of APCs is selected from exactly or about 1.5 x ¹⁰⁶ APCs/ ^cm2 The first gas permeable surface is seeded on the first gas permeable surface at a density in the range of exactly or about 3.5 x ¹⁰ APCs/cm ² , and in step (b), the second APC population is selected from the group consisting of exactly or about 3.5 x ¹⁰ APCs APC/cm ² to just or about 6.0×10 ⁶ APC/cm ² are seeded on the first gas permeable surface.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified wherein in step (a), the first population of APCs is selected from exactly or about 2.0 x ¹⁰⁶ APCs/ ^cm2 The first gas permeable surface is seeded on the first gas permeable surface at a density in the range of exactly or about 3.0 x ¹⁰ APCs/cm ² , and in step (b), the second APC population is selected from the group consisting of exactly or about 4.0 x ¹⁰ APCs APC/cm ² to just or about 5.5×10 ⁶ APC/cm ² are seeded on the first gas permeable surface.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable, modified wherein in step (a), the first APC population is present at a density of exactly or about 2.0 x ¹⁰⁶ APCs/ ^cm2 The first gas permeable surface is seeded, and in step (b), a second population of APCs is seeded on the first gas permeable surface at a density of just or about 4.0 x ¹⁰⁶ APCs/ ^cm2 .

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the APCs are peripheral blood mononuclear cells (PBMCs).

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the PBMCs are irradiated and exogenous to the donor of the first T cell population.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the T cells are tumor infiltrating lymphocytes (TILs).

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein the T cells are myeloid infiltrating lymphocytes (MIL).

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein the T cells are peripheral blood lymphocytes (PBL).

In other embodiments, the invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the first population of T cells is obtained by isolation from whole blood of a donor.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the first population of T cells is obtained by isolation from apheresis products of a donor.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the first T cell population is selected from whole blood or blood cells of a donor by positive or negative selection of T cell phenotype Separation product separation.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the T cell phenotype is CD3+ and CD45+.

In other embodiments, the invention provides a modification of the method described in any preceding paragraph as applicable above, wherein T cells are isolated from NK cells prior to performing the initial first expansion of the first T cell population. In other embodiments, T cells and NK cells in the first T cell population are separated by removing CD3-CD56+ cells from the first T cell population. In other embodiments, CD3-CD56+ cells are removed from the first T cell population by cell sorting the first T cell population using a confinement strategy that removes the CD3-CD56+ cell fraction and recovers the negative fraction. In other embodiments, the aforementioned methods are used for T cell expansion in a first T cell population characterized by a high percentage of NK cells. In other embodiments, the foregoing methods are used for T cell expansion in a first T cell population characterized by a high percentage of CD3-CD56+ cells. In other embodiments, the aforementioned methods are used for T cell expansion in tumor tissue characterized by the presence of high numbers of NK cells. In other embodiments, the aforementioned methods are used for T cell expansion in tumor tissue characterized by a large number of CD3-CD56+ cells. In other embodiments, the aforementioned methods are used for T cell expansion in tumor tissue obtained from a patient with a tumor characterized by the presence of a large number of NK cells. In other embodiments, the aforementioned methods are used for T cell expansion in tumor tissue obtained from a patient with a tumor characterized by the presence of large numbers of CD3-CD56+ cells. In other embodiments, the aforementioned methods are used for T cell expansion in tumor tissue obtained from a patient with ovarian cancer.

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified wherein exactly or about 1 x ¹⁰⁷ T cells from the first T cell population are seeded in the container to initially Initiation of the first expansion culture in such vessels.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified wherein the first population of T cells is distributed into a plurality of containers and each container is seeded with exactly or about 1 x ¹⁰⁷ T cells from the first T cell population are used to initiate the first expansion culture in such vessels.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified, wherein the second population of T cells collected in step (c) is a population of therapeutic TILs.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first T cell population is obtained from one or more mini biopsies of tumor tissue from a donor (including, for example, punched biopsies). biopsy), core needle biopsy, core needle biopsy, or fine needle aspirate.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsies of tumor tissue from donors (including, for example, punched biopsies). biopsy), core needle biopsy, core needle biopsy, or fine needle aspirate.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsies of tumor tissue from the donor (including, for example, punched biopsies). biopsy), core needle biopsy, core needle biopsy, or fine needle aspirate.

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tumor tissues from donors. needle aspirate.

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mini biopsies (including, for example, punch biopsies), core needle biopsies, core needle biopsies, or fine needle aspirates of tumor tissue from donors.

In other embodiments, the invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first T cell population is obtained from one or more core biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first T cell population is obtained from 1 to 20 biopsies of tumor tissue cores from donors.

In other embodiments, the invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first T cell population is obtained from 1 to 10 biopsies of tumor tissue cores from donors.

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 core needle biopsies of tumor tissue from donors.

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or Biopsy of 10 tumor tissue core needles from donors.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first population of T cells is obtained from one or more fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first T cell population is obtained from 1 to 20 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first population of T cells is obtained from 1 to 10 fine needle aspirates of tumor tissue from a donor.

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fine needle aspirates of tumor tissue from donors.

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fine-needle aspirates of tumor tissue from donors.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first T cell population is obtained from one or more mini biopsies of tumor tissue from a donor (including, for example, punched biopsies). inspection).

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsies of tumor tissue from donors (including, for example, punched biopsies). inspection).

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsies of tumor tissue from the donor (including, for example, punched biopsies). inspection).

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mini biopsies (including for example punch biopsies) of tumor tissue from the donor.

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mini biopsies (including eg punch biopsies) of tumor tissue from donors.

In other embodiments, the invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first population of T cells is obtained from one or more core biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first T cell population is obtained from 1 to 20 core needle biopsies of tumor tissue from a donor.

In other embodiments, the present invention provides a modification of the method described in any preceding paragraph as applicable, wherein the first T cell population is obtained from 1 to 10 core needle biopsies of tumor tissue from a donor.

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 core needle biopsies of tumor tissue from donors.

In other embodiments, the invention provides a method as described in any preceding paragraph as applicable, modified, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or Core needle aspiration biopsy of 10 tumor tissues from donors.

In other embodiments, the present invention provides methods for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: i) by culturing the tumor in a first cell culture medium comprising IL-2 Samples About 3 days to obtain and/or receive a first population of TILs from a tumor sample obtained from one or more mini-biopsies, core needle biopsies, or needle biopsies of tumors in an individual, and then from the second A TIL population to isolate CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL populations; (ii) by culturing CD39/ CD69 double-negative and/or CD39 ^LO /CD69 ^LO TIL populations are used for initial first amplification to produce a second TIL population, wherein the initial first amplification is carried out in a container comprising a first gas-permeable surface area, wherein Initiating the first expansion for a first period of about 7 or 8 days to obtain a second TIL population, wherein the number of the second TIL population is greater than the first TIL population; (iii) by using additional IL-2, OKT- 3 and APC supplementing the second cell culture medium of the second TIL population for rapid second expansion to produce a third TIL population, wherein the number of APCs added in the rapid second expansion is the number added in step (ii) At least twice the number of APCs, wherein the rapid second expansion is performed for a second period of about 11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is in a period comprising the second (iv) collecting the therapeutic TIL populations obtained from step (iii); and (v) transferring the collected TIL populations from step (iv) to an infusion bag.

In other embodiments, the present invention provides methods for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (i) by culturing in a first cell culture medium comprising IL-2 Tumor samples about 3 days to obtain and/or receive a first TIL population from a tumor sample obtained from one or more mini-biopsies, core biopsies, or needle biopsies of tumors in an individual, and then from The first TIL population isolates CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL populations; (ii) by culturing CD39 in a second cell culture medium containing IL-2, OKT-3 and antigen presenting cells (APCs) /CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL populations to perform an initial first expansion to generate a second TIL population, wherein the initial first expansion is performed for a first period of about 7 or 8 days to obtain the second TIL population Two TIL populations, wherein the second TIL population is greater in number than the first TIL population; (iii) rapid second expansion by contacting the second TIL population with a third cell culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the rapid second expansion is performed for a second period of about 11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population; and (iv) collected from step (iii ) obtained therapeutic TIL population.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified wherein after day 5 of the second period of time, the culture is divided into 2 or more subcultures, and Each subculture was supplemented with an additional amount of the third medium and grown for about 6 days.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified wherein after day 5 of the second period of time, the culture is divided into 2 or more subcultures, and Each subculture was supplemented with a fourth medium containing IL-2 and grown for about 6 days.

In other embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified wherein after day 5 of the second period of time, the culture is divided into up to 5 subcultures.

In other embodiments, the present invention provides the method described in any preceding paragraph as applicable above, modified, wherein all steps in the method are completed within about 22 days.

In other embodiments, the present invention provides a method for expanding T cells, comprising: (i) priming the first T cell population from a tumor sample by culturing the first T cell population; expanding, to achieve growth and to initiate activation of a first T cell population, the tumor sample is obtained from one or more mini biopsies, core needle biopsies or needle biopsies of the donor tumor; (ii) in step (a) After activation of the primed first T cell population begins to decline, a rapid second expansion of the first T cell population is achieved by culturing the first T cell population to achieve growth and enhance activation of the first T cell population to obtain a second a population of T cells; and (iv) collecting a second population of T cells. In some embodiments, the tumor sample is obtained from a plurality of core needle biopsies. In some embodiments, the plurality of core needle biopsies is selected from the group consisting of: 2, 3, 4, 5, 6, 7, 8, 9, and 10 core needle biopsies.

In some embodiments, the present invention provides methods described in any preceding paragraph as applicable, modified such that the first, second and/or third TIL populations are sorted for: (a) CD39/CD69 double negative And/or CD39 ^LO /CD69 ^LO , (b) CD39/CD69 double knockout, or (c) a combination of (a) and (b) TIL. Cell sorting systems suitable for sorting TILs according to the methods of the invention can be found in US Application No. 2019/0212332: (a) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO , (b) CD39 /CD69 double knockout, or a combination of (i) and (ii), which are incorporated herein by reference. In some embodiments, cells are sorted using the method described in Zhang, X et al., "High Throughput Cell Sorting by Surface Free Energy Activation", "Analytical Chemistry" (2014), 86: 9350-9355. In some embodiments, the cell sorting technology has a capacity of 5e7 cells per second. In some embodiments, a capacity of 5e7 cells per second can be used for sorting at day 22 (eg, after expansion of TILs). In some embodiments, the invention provides therapeutic TIL populations or TIL compositions expanded using 1-REP or 2-REP protocols (see Example 15 and Figure 41). In some embodiments, TIL populations will be assessed for growth, survival, phenotype, function, autologous tumor killing, and TCRvβ repertoire. In some embodiments, a portion of a therapeutic TIL population or TIL composition can be sampled for testing using the cell sorting methods described above.

In some embodiments, the invention provides the method described in any preceding paragraph as applicable above, modified such that T cells or TILs are obtained from tumor digests. In some embodiments, tumors are grown by incubating in an enzyme medium such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamycin, 30 U/mL deoxyribonuclease, and 1.0 mg/mL collagenase , followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA) to generate tumor digests. In some embodiments, the tumor is placed in a tumor dissociating enzyme mixture comprising one or more dissociation (digesting) enzymes such as but not limited to collagenase (including any blend or type of collagenase) , Accutase™, Accumax™, hyaluronidase, dispase (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, protease type XIV (pronase), deoxygenated Ribonuclease I (DNase), trypsin inhibitors, any other dissociative or proteolytic enzymes, and any combination thereof. In other embodiments, the tumor is placed in a tumor dissociating enzyme mixture comprising collagenase (including any blend or type of collagenase), neutral protease (dispase), and DNA Enzyme I (DNase). VI. Pharmaceutical Compositions, Dosages and Administration Regimen

In some embodiments, TILs, MILs or PBLs expanded and/or genetically modified using the methods of the present disclosure, including TILs, MILs or PBLs genetically modified to express CCR, are administered to patients as pharmaceutical compositions. In some embodiments, the pharmaceutical composition is a suspension of TIL in a sterile buffer. TILs expanded using the PBMCs of the disclosure can be administered by any suitable route known in the art. In some embodiments, T cells are administered as a single intraarterial or intravenous infusion, which preferably lasts for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

Any suitable dose of TIL can be administered. In some embodiments, about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ TILs are administered, with an average of about 7.8×10 ¹⁰ TILs, particularly where the cancer is NSCLC or melanoma. In some embodiments, about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ TILs are administered. In some embodiments, about 3×10 ¹⁰ to about 12×10 ¹⁰ TILs are administered. In some embodiments, about 4×10 ¹⁰ to about 10×10 ¹⁰ TILs are administered. In some embodiments, about 5×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, about 6×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, about 7×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is from about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ . In some embodiments, the therapeutically effective dose is about 7.8 x ¹⁰¹⁰ TILs, especially when the cancer is melanoma. In some embodiments, the therapeutically effective dose is about 7.8×10 ¹⁰ TILs, especially when the cancer is NSCLC. In some embodiments, the therapeutically effective dose is from about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3×10 ¹⁰ to about 12×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 4×10 ¹⁰ to about 10×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 5×10 ¹⁰ to about 8×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 6×10 ¹⁰ to about 8×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7×10 ¹⁰ to about 8×10 ¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical composition of the invention is about 1×10 ⁶ , 2×10 6 , 3×10 ⁶ , 4×10 ⁶ , 5×10 ^{6 ,} 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ^{11 , 5×10 11 , 6×10 11 ,} 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some embodiments, the number of TILs provided in the pharmaceutical composition of the present invention is 1×10 ⁶ to 5×10 ⁶ , 5×10 ⁶ to 1×10 ⁷ , 1×10 ⁷ to 5×10 ⁷ , 5×10 ⁷ to 1×10 ⁸ , 1×10 ⁸ to 5×10 ⁸ , 5×10 ⁸ to 1×10 ⁹ , 1×10 ⁹ to 5×10 ⁹ , 5×10 ⁹ to 1×10 ¹⁰ , 1×10 ¹⁰ to 5×10 ¹⁰ , 5×10 ¹⁰ to 1×10 ¹¹ , 5×10 ¹¹ to 1×10 ¹² , 1×10 ¹² to 5×10 ¹² and 5×10 ¹² to 1×10 ¹³ within range.

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% of the pharmaceutical composition. %, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% , 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002 % or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75% of the pharmaceutical composition , 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50 %, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75%, 7.50%, 7.25% , 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3 %, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008% , 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, TIL is provided in the pharmaceutical composition of the present invention at a concentration of about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% of the pharmaceutical composition % to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to About 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17% %, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v or v /v range.

In some embodiments, TIL is provided in the pharmaceutical composition of the present invention at a concentration of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% of the pharmaceutical composition % to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to In the range of about 1%, about 0.1% to about 0.9% w/w, w/v or v/v.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g , 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g or 0.0001 g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the invention is greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g , 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g or 10 g.

The TILs provided in the pharmaceutical compositions of the present invention are effective over a wide dosage range. The exact dosage will depend on the route of administration, the form of compound administration, the sex and age of the individual to be treated, the weight of the individual to be treated, and the preference and experience of the attending physician. Clinically determined doses of TIL can also be used as appropriate. The amount of pharmaceutical composition administered using the methods herein, such as the dose of TIL, will depend on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the formulation of the active pharmaceutical ingredient, and the judgment of the prescribing physician. depends.

In some embodiments, TILs can be administered in a single dose. Such administration can be by injection (eg, intravenous injection). In some embodiments, TILs can be administered in multiple doses. Dosing may be once, twice, three, four, five, six, or more than six times per year. Dosing can be monthly, biweekly, weekly, or every other day. Administration of TILs can continue as desired.

In some embodiments, the effective dose of TIL is about 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 6 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 8 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ^{11 , 6×10 11} , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2× ^{10 12 ,} 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ^{12 , 7×10 12 ,} 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some embodiments, the effective dose of TIL is 1×10 ⁶ to 5×10 ⁶ , 5×10 ⁶ to 1×10 ⁷ , 1×10 ⁷ to 5×10 ⁷ , 5×10 ⁷ to 1×10 ⁸ , 1×10 ⁸ to 5×10 ⁸ , 5×10 ⁸ to 1×10 ⁹ , 1×10 ⁹ to 5×10 ⁹ , 5×10 ⁹ to 1×10 ¹⁰ , 1×10 ¹⁰ to 5×10 ¹⁰ , 5×10 ¹⁰ to 1×10 ¹¹ , 5×10 ¹¹ to 1×10 ¹² , 1×10 ¹² to 5×10 ¹² and 5×10 ¹² to 1×10 ¹³ .

In some embodiments, the effective dosage of TIL is about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg /kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg /kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg , about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, About 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dosage of TIL is about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg , about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg range.

Effective amounts of TIL can be administered by any of the recognized modes of administration of agents of similar utility, including intranasal and transdermal routes, by intraarterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical , by implantation or by inhalation, administered in single or multiple doses.

In other embodiments, the invention provides an infusion bag comprising a therapeutic TIL population as described in any of the preceding paragraphs above.

In other embodiments, the present invention provides a tumor infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population as described above in any preceding paragraph and a pharmaceutically acceptable carrier.

In other embodiments, the present invention provides an infusion bag comprising a TIL composition as described above in any preceding paragraph.

In other embodiments, the invention provides a cryopreserved formulation of a therapeutic TIL population as described above in any preceding paragraph.

In other embodiments, the present invention provides a tumor infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population as described above in any preceding paragraph and a cryopreservation medium.

In other embodiments, the invention provides a TIL composition as described in any preceding paragraph above, modified wherein the cryopreservation medium comprises DMSO.

In other embodiments, the invention provides a modified TIL composition as described in any preceding paragraph, wherein the cryopreservation medium contains 7-10% DMSO.

In other embodiments, the invention provides a cryopreserved formulation of a TIL composition as described above in any preceding paragraph.

In some embodiments, TILs expanded using the methods of the disclosure are administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TIL in a sterile buffer. TILs expanded using the PBMCs of the disclosure can be administered by any suitable route known in the art. In some embodiments, T cells are administered as a single intraarterial or intravenous infusion, which preferably lasts for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

Any suitable dose of TIL can be administered. In some embodiments, about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ TILs are administered, with an average of about 7.8×10 ¹⁰ TILs, particularly where the cancer is NSCLC. In some embodiments, about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ TILs are administered. In some embodiments, about 3×10 ¹⁰ to about 12×10 ¹⁰ TILs are administered. In some embodiments, about 4×10 ¹⁰ to about 10×10 ¹⁰ TILs are administered. In some embodiments, about 5×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, about 6×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, about 7×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is from about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ . In some embodiments, the therapeutically effective dose is about 7.8 x ¹⁰¹⁰ TILs, especially where the cancer is melanoma. In some embodiments, the therapeutically effective dose is from about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3×10 ¹⁰ to about 12×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 4×10 ¹⁰ to about 10×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 5×10 ¹⁰ to about 8×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is from about 6×10 ¹⁰ to about 8×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7×10 ¹⁰ to about 8×10 ¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical composition of the invention is about 1×10 ⁶ , 2×10 6 , 3×10 ⁶ , 4×10 ⁶ , 5×10 ^{6 ,} 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ^{11 , 5×10 11 , 6×10 11 ,} 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some embodiments, the number of TILs provided in the pharmaceutical composition of the present invention is 1×10 ⁶ to 5×10 ⁶ , 5×10 ⁶ to 1×10 ⁷ , 1×10 ⁷ to 5×10 ⁷ , 5×10 ⁷ to 1×10 ⁸ , 1×10 ⁸ to 5×10 ⁸ , 5×10 ⁸ to 1×10 ⁹ , 1×10 ⁹ to 5×10 ⁹ , 5×10 ⁹ to 1×10 ¹⁰ , 1×10 ¹⁰ to 5×10 ¹⁰ , 5×10 ¹⁰ to 1×10 ¹¹ , 5×10 ¹¹ to 1×10 ¹² , 1×10 ¹² to 5×10 ¹² and 5×10 ¹² to 1×10 ¹³ within range.

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% of the pharmaceutical composition. %, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% , 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002 % or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75% of the pharmaceutical composition , 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50 %, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75%, 7.50%, 7.25% , 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3 %, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008% , 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, TIL is provided in the pharmaceutical composition of the present invention at a concentration of about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% of the pharmaceutical composition % to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to About 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17% %, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v or v /v range.

In some embodiments, TIL is provided in the pharmaceutical composition of the present invention at a concentration of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% of the pharmaceutical composition % to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to In the range of about 1%, about 0.1% to about 0.9% w/w, w/v or v/v.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g , 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g or 0.0001 g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the invention is greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g , 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g or 10 g.

The TILs provided in the pharmaceutical compositions of the present invention are effective over a wide dosage range. The exact dosage will depend on the route of administration, the form of compound administration, the sex and age of the individual to be treated, the weight of the individual to be treated, and the preference and experience of the attending physician. Clinically determined doses of TIL can also be used as appropriate. The amount of pharmaceutical composition administered using the methods herein, such as the dose of TIL, will depend on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the formulation of the active pharmaceutical ingredient, and the judgment of the prescribing physician. depends.

In some embodiments, TILs can be administered in a single dose. Such administration can be by injection (eg, intravenous injection). In some embodiments, TILs can be administered in multiple doses. Dosing may be once, twice, three, four, five, six, or more than six times per year. Dosing can be monthly, biweekly, weekly, or every other day. Administration of TILs can continue as desired.

In some embodiments, the effective dose of TIL is about 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 6 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ^{7 , 3×10 7 ,} 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 8 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ^{11 , 6×10 11} , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2× ^{10 12 ,} 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ^{12 , 7×10 12 ,} 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some embodiments, the effective dose of TIL is 1×10 ⁶ to 5×10 ⁶ , 5×10 ⁶ to 1×10 ⁷ , 1×10 ⁷ to 5×10 ⁷ , 5×10 ⁷ to 1×10 ⁸ , 1×10 ⁸ to 5×10 ⁸ , 5×10 ⁸ to 1×10 ⁹ , 1×10 ⁹ to 5×10 ⁹ , 5×10 ⁹ to 1×10 ¹⁰ , 1×10 ¹⁰ to 5×10 ¹⁰ , 5×10 ¹⁰ to 1×10 ¹¹ , 5×10 ¹¹ to 1×10 ¹² , 1×10 ¹² to 5×10 ¹² and 5×10 ¹² to 1×10 ¹³ .

In some embodiments, the effective dosage of TIL is about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg /kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg /kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg , about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, About 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose of TIL is from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg , about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg range.

Effective amounts of TIL can be administered by any of the recognized modes of administration of agents of similar utility, including intranasal and transdermal routes, by intraarterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical , by implantation or by inhalation, administered in single or multiple doses. VII. Methods of Treating Patients

The therapeutic approach begins with primary TIL collection and TIL culture. Such methods have been described in the field, eg, Jin et al., Journal of Immunotherapy, 2012 , 35(3):283-292, which is hereby incorporated by reference in its entirety. Embodiments of methods of treatment are described below throughout the various sections, including the Examples.

Especially suitable according to the methods described herein are amplified TILs comprising, for example, those described above in Steps A to F or produced according to Steps A to F above (also shown in, for example, FIG. 1 and/or FIG. 8 ). Described in treating cancer patients (e.g., Goff et al., J. Clinical Oncology , 2016 , 34(20):2389-239 and supplements; incorporated herein by reference in its entirety ). In some embodiments, TILs are grown from resected metastatic melanoma deposits as previously described (see Dudley et al., J Immunotherapeutics, 2003 , 26:332-342; which is incorporated by reference in its entirety incorporated into this article). Fresh tumors can be dissected under sterile conditions. Representative samples may be collected for formal pathological analysis. Individual fragments of 2 mm ³ to 3 mm ³ can be used. In some embodiments, 5, 10, 15, 20, 25 or 30 samples are obtained from each patient. In some embodiments, 20, 25 or 30 samples are obtained from each patient. In some embodiments, 20, 22, 24, 26 or 28 samples are obtained from each patient. In some embodiments, 24 samples are obtained from each patient. Samples can be placed in individual wells of a 24-well plate, maintained in growth medium containing high doses of IL-2 (6,000 IU/mL), and monitored for tumor destruction and/or TIL proliferation. Any tumor enzymes remaining in viable cells after treatment can be dissociated into a single cell suspension and stored frozen, as described herein.

In some embodiments, successfully grown TILs can be sampled for phenotypic analysis (CD3, CD4, CD8, and CD56) and tested against autologous tumors when available. TILs were considered reactive if overnight co-cultures produced interferon-γ (IFN-γ) levels > 200 pg/mL and double background. (Goff et al., J Immunotherapeutics, 2010 , 33:840-847; incorporated herein by reference in its entirety). In some embodiments, cultures demonstrating self-reactivity or a sufficient growth pattern can be selected for a second expansion (e.g., according to the second expansion provided in step D of FIG. 1 and/or FIG. 8 ). ), including a second amplification sometimes referred to as rapid amplification (REP). In some embodiments, expanded TILs that are highly self-reactive (eg, highly proliferative during the second expansion) are selected for an additional second expansion. In some embodiments, TILs with high autoreactivity (e.g., high proliferation during the second expansion as provided in step D of FIG. 1 and/or FIG. 8 ) are selected for use in accordance with FIG. 1 and/or FIG. Or the additional second amplification of step D of FIG. 8 .

Cryopreserved samples of infusion bag TIL can be analyzed by flow cytometry (eg FlowJo) (BD Biosciences) for the surface markers CD3, CD4, CD8, CCR7 and CD45RA and by any of the methods described herein. cell phenotype. Serum cytokines were measured by using standard enzyme-linked immunosorbent assay techniques. An increase in serum IFN-g was defined as >100 pg/mL and greater than a baseline level of 43.

In some embodiments, TILs produced by the methods provided herein, eg, the methods exemplified in FIG. 1 and/or FIG. 8 , achieve a surprising improvement in the clinical efficacy of TILs. In some embodiments, compared to TILs produced by methods other than those described herein (including, for example, methods other than those illustrated in FIG. 1 and/or in FIG. 8 ), by the methods provided herein Methods such as those exemplified in Figure 1 and/or Figure 8 produce TILs exhibiting improved clinical efficacy. In some embodiments, methods other than those described herein include methods referred to as Process 1C and/or Gen 1 . In some embodiments, increased efficacy is measured by DCR, ORR, and/or other clinical response measures. In some embodiments, TILs produced by methods provided herein are compared to TILs produced by methods other than those described herein (including, for example, methods other than those illustrated in FIG. 1 and/or FIG. 8 ). , TILs produced by methods such as those exemplified in Figure 1 or Figure 8 exhibited similar response times and safety profiles.

In some embodiments, IFN-γ is indicative of therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of an individual treated with TIL is indicative of active TIL. In some embodiments, potency assays for IFN-γ production are employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by measuring the level of cytokine IFN-γ in the blood, serum or ex vivo TIL of individuals treated with TIL prepared by the method of the present invention, such methods include, for example, FIG. 1 and/or FIG. The method described in 8. In some embodiments, an increase in IFN-γ is indicative of therapeutic efficacy in patients treated with TILs produced by the methods of the invention. In some embodiments, compared to untreated patients and/or compared to those prepared using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ), In TIL-treated patients, IFN-γ doubled, doubled, tripled, quadrupled, or fivefold or more. In some embodiments, compared to untreated patients and/or compared to those prepared using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ), In TIL-treated patients, IFN-γ secretion doubled. In some embodiments, compared to untreated patients and/or compared to those prepared using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ), In TIL-treated patients, IFN-γ secretion increased two-fold. In some embodiments, compared to untreated patients and/or compared to TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ) In treated patients, IFN-γ secretion increased three-fold. In some embodiments, compared to untreated patients and/or compared to TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ) In treated patients, IFN-γ secretion increased four-fold. In some embodiments, compared to untreated patients and/or compared to TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ) In treated patients, IFN-γ secretion increased five-fold. In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ is measured in ex vivo TILs of individuals treated with TILs prepared by the methods of the invention, including, for example, the methods described in FIG. 1 and/or FIG. 8 . In some embodiments, IFN-γ is measured in the blood of subjects treated with TILs prepared by the methods of the invention, including, for example, the methods described in FIG. 1 and/or FIG. 8 . In some embodiments, IFN-γ is measured in the serum of TILs of individuals treated with TILs prepared by the methods of the invention, including, for example, the methods described in FIG. 1 and/or FIG. 8 . In some embodiments, IFN-γ (IFN-gamma) is indicative of therapeutic efficacy and/or increased clinical efficacy in cancer treatment.

In some embodiments, TILs prepared by the methods of the invention include TILs as depicted in, eg, FIG. 1 or FIG. 8 . In some embodiments, IFN-γ is indicative of therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of an individual treated with TIL is indicative of active TIL. In some embodiments, potency assays for IFN-γ production are employed. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by measuring the level of cytokine IFN-γ in the blood, serum or ex vivo TIL of individuals treated with TIL prepared by the method of the present invention, such methods include, for example, FIG. 1 and/or FIG. The method described in 8. In some embodiments, an increase in IFN-γ is indicative of therapeutic efficacy in patients treated with TILs produced by the methods of the invention. In some embodiments, compared to untreated patients and/or compared to prepared using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ), In TIL-treated patients, IFN-γ doubled, doubled, tripled, quadrupled, or fivefold or more.

In some embodiments, compared to TIL produced by other methods (including methods not illustrated in FIG. 1 and/or FIG. TILs prepared as, for example, the methods described in Figure 1 and/or Figure 8) exhibit increased pluripotency. In some embodiments, significantly increased polyclonality and/or increased polyclonality is indicative of therapeutic efficacy and/or increased clinical efficacy. In some embodiments, polyclonality refers to T cell repertoire diversity. In some embodiments, increased polyclonality can be indicative of therapeutic efficacy with respect to administration of TILs produced by the methods of the invention. In some embodiments, pluripotency is doubled, doubled, compared to TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ). times, ten times, 100 times, 500 times or 1000 times. In some embodiments, compared to untreated patients and/or compared to using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ) In patients treated with TIL, polyclonality was doubled. In some embodiments, compared to untreated patients and/or compared to using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ) In patients treated with TIL, polyclonality was doubled. In some embodiments, compared to untreated patients and/or compared to using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ) In patients treated with TIL, polyclonality increased tenfold. In some embodiments, compared to untreated patients and/or compared to using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ) In patients treated with TIL, polyclonality increased 100-fold. In some embodiments, compared to untreated patients and/or compared to using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ) In patients treated with TIL, polyclonality increased 500-fold. In some embodiments, compared to untreated patients and/or compared to using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 and/or FIG. 8 ) In patients treated with TIL, polyclonality increased 1000-fold.

Measures of efficacy can include disease control rate (DCR) and overall response rate (ORR), as known in the art and described herein. A. Methods of treating cancer

The compositions and methods described herein can be used in a method of treating disease. In some embodiments, it is used to treat a hyperproliferative disorder, such as cancer, in an adult patient or a pediatric patient. It can also be used to treat other disorders as described herein and in the following paragraphs.

In some embodiments, the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triple negative breast cancer), bone cancer, cancers caused by human papillomavirus (HPV), central nervous system Systemic cancers (including ependymoma, medulloblastoma, neuroblastoma, pinealoblastoma, and primitive neuroectodermal tumor), cervical cancer (including squamous cell carcinoma of the cervix, adenosquamous cervical cancer and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, Head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), hypopharynx cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer and pharyngeal cancer), kidney cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer), melanoma (including uveal, choroidal, ciliary body, or iris melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic Cancer of the liver (including pancreatic duct adenocarcinoma), cancer of the penis, rectum, kidney, renal cell carcinoma, sarcoma (including Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma and other bone and soft tissue sarcomas), thyroid cancer (including degenerative thyroid cancer), uterine cancer and vaginal cancer.

In some embodiments, the hyperproliferative disorder is a hematologic malignancy. In some embodiments, the hematological malignancy is selected from the group consisting of chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma ), Hodgkin's lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma. In some embodiments, the invention includes a method of treating a patient with cancer, wherein the cancer is a hematological malignancy. In some embodiments, the invention includes a method of treating a patient with cancer using a TIL, MIL, or PBL modified to express one or more CCRs, wherein the cancer is a hematological malignancy. In some embodiments, the invention includes a method of treating a patient with cancer using a MIL or PBL modified to express one or more CCRs, wherein the cancer is a hematological malignancy.

In some embodiments, the cancer is one of the aforementioned cancers, including solid tumor cancers and hematological malignancies, that is relapsed or refractory to treatment with at least one prior therapy, including chemotherapy, radiation therapy, or immunotherapy. In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least two prior therapies, including chemotherapy, radiation therapy, and/or immunotherapy. In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least three prior therapies, including chemotherapy, radiation therapy, and/or immunotherapy.

In some embodiments, the cancer is microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) cancer. MSI-H and dMMR cancers and their tests have been described in Kawakami et al., Curr. Treat. Options Oncol . 2015, 16, 30, the disclosures of which are incorporated herein by reference .

In some embodiments, the invention includes a method of treating a patient with cancer using a TIL, MIL, or PBL modified to express one or more CCRs, wherein the patient is human. In some embodiments, the invention includes a method of treating a patient with cancer using TIL, MIL, or PBL modified to express one or more CCRs, wherein the patient is non-human. In some embodiments, the invention includes a method of treating a patient with cancer using a TIL, MIL, or PBL modified to express one or more CCRs, wherein the patient is a companion animal.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the cancer is refractory to BRAF inhibitors and/or MEK inhibitors. In some embodiments, the present invention includes a method of treating a patient with cancer, wherein the cancer is refractory to treatment with a BRAF inhibitor selected from the group consisting of: vemurafenib, dabrafenib, enrafil Nil, Sorafenib and their pharmaceutically acceptable salts or solvates. In some embodiments, the invention includes a method of treating a patient with cancer, wherein the cancer is refractory to treatment with a MEK inhibitor selected from the group consisting of trametinib, cobimetinib, bimetinib Pimasertinib, selumetinib, pimasertinib, rifatinib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention includes a method of treating a patient with cancer, wherein the cancer is refractory to treatment with a BRAF inhibitor selected from the group consisting of: vemurafenib, dabrafenib, enrafil Ni, sorafenib, and pharmaceutically acceptable salts or solvates thereof; and refractory to treatment with MEK inhibitors selected from the group consisting of trametinib, cobimetinib, bametinib , selumetinib, pimacetinib, rifatinib and their pharmaceutically acceptable salts or solvates.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the cancer is a pediatric cancer.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the cancer is uveal melanoma.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the uveal melanoma is choroidal melanoma, ciliary body melanoma, or iris melanoma.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the pediatric cancer is neuroblastoma.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the pediatric cancer is sarcoma.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the sarcoma is osteosarcoma.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the sarcoma is a soft tissue sarcoma.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the soft tissue sarcoma is rhabdomyosarcoma, Ewing's sarcoma, or primitive neuroectodermal tumor (PNET).

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the pediatric cancer is a central nervous system (CNS) related cancer. In some embodiments, the pediatric cancer is refractory to chemotherapy. In some embodiments, the pediatric cancer is refractory to radiation therapy. In some embodiments, the pediatric cancer is refractory to treatment with dinutuximab.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the CNS-related cancer is medulloblastoma, pinealoblastoma, glioma, ependymoma, or glioblastoma tumor.

The compositions and methods described herein can be used in methods of treating cancer where the cancer is refractory to or resistant to prior treatment with an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the patient is a primary patient refractory to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient has not demonstrated a prior response to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient demonstrates a prior response to an anti-PD-1 or anti-PD-L1 antibody, followed by progression of the patient's cancer. In some embodiments, the cancer is refractory to treatment with a combination of an anti-CTLA-4 antibody and/or an anti-PD-1 or anti-PD-L1 antibody and at least one chemotherapeutic agent. In some embodiments, the prior chemotherapeutic agent is carboplatin, paclitaxel, pemetrexed, and/or cisplatin. In some of the previous embodiments, the chemotherapeutic agent is a platinum doublet chemotherapeutic agent. In some embodiments, the platinum dual therapy comprises a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin, and a chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine, and taxanes (including, for example, paclitaxel). (paclitaxel), docetaxel (docetaxel) or albumin-bound paclitaxel (nab-paclitaxel)) as a second chemotherapeutic agent. In some embodiments, a platinum doublet chemotherapeutic is combined with pemetrexed.

In some embodiments, the NSCLC is PD-L1 negative and/or is from a patient with a cancer expressing PD-L1 with a Tumor Proportion Score (TPS) <1%, as described elsewhere herein.

In some embodiments, the NSCLC is refractory to treatment with a combination therapy comprising an anti-PD-1 antibody or an anti-PD-L1 antibody and a platinum doublet therapy, wherein the platinum doublet therapy comprises: i) a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin, ii) and a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine and taxanes (including eg paclitaxel, docetaxel or nab-paclitaxel).

In some embodiments, the NSCLC is refractory to a combination therapy comprising an anti-PD-1 antibody or an anti-PD-L1 antibody, pemetrexed, and a platinum doublet therapy, wherein the platinum doublet therapy comprises: i) a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin, ii) and a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine and taxanes (including eg paclitaxel, docetaxel or nab-paclitaxel).

In some embodiments, the NSCLC has been treated with an anti-PD-1 antibody. In some embodiments, the NSCLC has been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC patient is treatment naïve. In some embodiments, the NSCLC has not been treated with an anti-PD-1 antibody. In some embodiments, the NSCLC has not been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC has been previously treated with a chemotherapeutic agent. In some embodiments, the NSCLC has been previously treated with a chemotherapeutic agent, but is no longer being treated with that chemotherapeutic agent. In some embodiments, the NSCLC patient is not receiving anti-PD-1/PD-L1 therapy. In some embodiments, the NSCLC patient has low PD-L1 expression. In some embodiments, the NSCLC patient is NSCLC naïve or after chemotherapeutic treatment but not anti-PD-1/PD-L1 therapy. In some embodiments, the NSCLC patient is treatment naïve or treated with a chemotherapeutic agent but not anti-PD-1/PD-L1 and has low PD-L1 expression. In some embodiments, the NSCLC patient has macroscopic disease at baseline. In some embodiments, the individual has macroscopic disease with low PD-L1 expression at baseline. In some embodiments, the NSCLC patient has no detectable expression of PD-L1. In some embodiments, the NSCLC patient is treatment naïve or treated with a chemotherapeutic agent but naïve to anti-PD-1/PD-L1 therapy and has no detectable expression of PD-L1. In some embodiments, the patient has macroscopic disease and no detectable expression of PD-L1 at baseline. In some embodiments, the NSCLC patient has untreated NSCLC or is on chemotherapy (e.g., after a chemotherapeutic agent) without anti-PD-1/PD-L1 treatment, and the patient has low PD-L1 expression and /or have macroscopic disease at baseline. In some embodiments, macrofocal disease is indicated when the largest tumor is greater than 7 cm in diameter as measured in the transverse or coronal plane. In some embodiments, macrofocal disease is indicated when there are swollen lymph nodes with a short axis diameter of 20 mm or greater. In some embodiments, chemotherapeutic agents include standard-of-care treatments for NSCLC.

In some embodiments, PD-L1 expression is determined by tumor proportion score. In some embodiments, individuals with refractory NSCLC tumors have a Tumor Proportion Score (TPS) of <1%. In some embodiments, individuals with refractory NSCLC tumors have TPS > 1%. In some embodiments, the individual with refractory NSCLC has previously been treated with an anti-PD-1 antibody and/or an anti-PD-L1 antibody and the tumor proportion score is based on the anti-PD-1 antibody and/or anti-PD-L1 antibody determined prior to treatment. In some embodiments, the individual with refractory NSCLC has been previously treated with an anti-PD-L1 antibody and the tumor proportion score is determined prior to the anti-PD-L1 antibody treatment.

In some embodiments, compared to TILs produced by other methods (including methods not illustrated in FIG. 1 or the method described in Figure 8) exhibited increased pluripotency. In some embodiments, significantly increased polyclonality and/or increased polyclonality is indicative of therapeutic efficacy and/or increased clinical efficacy for cancer therapy. In some embodiments, polyclonality refers to T cell repertoire diversity. In some embodiments, increased polyclonality can be indicative of therapeutic efficacy with respect to administration of TILs produced by the methods of the invention. In some embodiments, compared to TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in FIG. 1 or FIG. Ten times, 100 times, 500 times or 1000 times. In some embodiments, compared to untreated patients and/or compared to treatment with TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in Figure 1 or Figure 8 ) In patients, polyclonality doubles. In some embodiments, compared to untreated patients and/or compared to treatment with TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in Figure 1 or Figure 8 ) In patients, polyclonality doubled. In some embodiments, compared to untreated patients and/or compared to treatment with TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in Figure 1 or Figure 8 ) In patients with multiple strains, the polyclonality increased tenfold. In some embodiments, compared to untreated patients and/or compared to treatment with TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in Figure 1 or Figure 8 ) In patients, polyclonality increased 100-fold. In some embodiments, compared to untreated patients and/or compared to treatment with TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in Figure 1 or Figure 8 ) In patients, polyclonality increased 500-fold. In some embodiments, compared to untreated patients and/or compared to treatment with TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in Figure 1 or Figure 8 ) In patients, polyclonality increased 1000-fold.

In some embodiments, PD-L1 expression is determined by tumor proportion score using one or more assays as described herein. In some embodiments, an individual or patient with a NSCLC tumor has a Tumor Proportion Score (TPS) of <1%. In some embodiments, the NSCLC tumor has TPS > 1%. In some embodiments, the individual or patient with NSCLC has been previously treated with an anti-PD-1 antibody and/or an anti-PD-L1 antibody and the tumor proportion score is based on the anti-PD-1 antibody and/or anti-PD-L1 antibody determined prior to treatment. In some embodiments, the individual or patient with NSCLC has been previously treated with an anti-PD-L1 antibody and the tumor proportion score is determined prior to the anti-PD-L1 antibody treatment. In some embodiments, individuals or patients with refractory or drug-resistant NSCLC tumors have a Tumor Proportion Score (TPS) of <1%. In some embodiments, individuals or patients with refractory or drug-resistant NSCLC tumors have TPS > 1%. In some embodiments, the individual or patient with refractory or drug-resistant NSCLC has previously been treated with an anti-PD-1 antibody and/or an anti-PD-L1 antibody and the tumor proportion score is based on the anti-PD-1 antibody and/or anti-PD-L1 antibody Or determined before anti-PD-L1 antibody therapy. In some embodiments, the individual or patient with refractory or drug-resistant NSCLC has been previously treated with an anti-PD-L1 antibody and the tumor proportion score is determined prior to such anti-PD-L1 antibody treatment.

In some embodiments, the NSCLC exhibits a Tumor Proportion Score (TPS), or the percentage of viable tumor cells taken from a patient prior to anti-PD-1 or anti-PD-L1 therapy, for NSCLC at any intensity indicated for PD-L1 protein Less than 1% (TPS < 1%) partial or complete membrane staining. In some embodiments, the NSCLC is a NSCLC exhibiting a TPS selected from the group consisting of: <50%, <45%, <40%, <35%, <30%, <25%, <20%, < 15%, <10%, <9%, <8%, <7%, <6%, <5%, <4%, <3%, <2%, <1%, <0.9%, <0.8% , <0.7%, <0.6%, <0.5%, <0.4%, <0.3%, <0.2%, <0.1%, <0.09%, <0.08%, <0.07%, <0.06%, <0.05%, < 0.04%, <0.03%, <0.02% and <0.01%. In some embodiments, the NSCLC is a NSCLC exhibiting a TPS selected from the group consisting of about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8% , about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, approximately 0.03%, approximately 0.02% and approximately 0.01%. In some embodiments, the NSCLC is a NSCLC exhibiting a TPS between 0% and 1%. In some embodiments, the NSCLC is an NSCLC exhibiting a TPS between 0% and 0.9%. In some embodiments, the NSCLC is an NSCLC exhibiting a TPS between 0% and 0.8%. In some embodiments, the NSCLC is a NSCLC exhibiting a TPS between 0% and 0.7%. In some embodiments, the NSCLC is a NSCLC exhibiting a TPS between 0% and 0.6%. In some embodiments, the NSCLC is a NSCLC exhibiting a TPS between 0% and 0.5%. In some embodiments, the NSCLC is a NSCLC exhibiting a TPS between 0% and 0.4%. In some embodiments, the NSCLC is a NSCLC exhibiting a TPS between 0% and 0.3%. In some embodiments, the NSCLC is a NSCLC exhibiting a TPS between 0% and 0.2%. In some embodiments, the NSCLC is a NSCLC exhibiting a TPS between 0% and 0.1%. TPS can be measured by methods known in the art, such as those described in Hirsch et al., J. Thorac. Oncol. 2017 , 12 , 208-222 or for use in A method for measuring TPS prior to treatment with pembrolizumab or other anti-PD-1 or anti-PD-L1 therapy. Methods approved by the US Food and Drug Administration for measuring TPS can also be used. In some embodiments, the PD-L1 is exosomal PD-L1. In some embodiments, PD-L1 is found on circulating tumor cells.

In some embodiments, partial membrane staining includes 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% %, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or higher percentages. In some embodiments, complete membrane staining includes about 100% membrane staining.

In some embodiments, the test for PD-L1 may involve measuring the level of PD-L1 in the patient's serum. In these embodiments, the measurement of PD-L1 in the patient's serum eliminates the uncertainty of tumor heterogeneity and the discomfort of serial biopsies performed by the patient.

In some embodiments, elevated soluble PD-L1 compared to baseline or normative levels is associated with prognosis of progression of NSCLC. See eg Okuma et al., Clinical Lung Cancer, 2018 , 19 , 410-417; Vecchiarelli et al., Oncotarget , 2018 , 9 , 17554-17563. In some embodiments, the PD-L1 is exosomal PD-L1. In some embodiments, PD-L1 is expressed on circulating tumor cells.

In some embodiments, the present invention provides a method of treating non-small cell lung cancer (NSCLC) by administering a population of tumor infiltrating lymphocytes (TILs) to an individual or patient in need thereof, wherein the individual or patient has at least one of: PD-L1 predetermined tumor proportion score (TPS) <1%, The TPS score of PD-L1 is 1%-49%, or Predetermined deletion of one or more driver mutations, wherein the driver mutation is selected from the group consisting of: EGFR mutation, EGFR insertion, EGFR exon 20 mutation, KRAS mutation, BRAF mutation, ALK mutation, c-ROS mutation (ROS1 mutation), ROS1 fusion, RET mutation, RET fusion , ERBB2 mutation, ERBB2 amplification, BRCA mutation, MAP2K1 mutation, PIK3CA, CDKN2A, PTEN mutation, UMD mutation, NRAS mutation, KRAS mutation, NF1 mutation, MET mutation, MET splicing and/or altered MET signaling, TP53 mutation, CREBBP mutation, KMT2C mutation, KMT2D mutation, ARID1A mutation, RB1 mutation, ATM mutation, SETD2 mutation, FLT3 mutation, PTPN11 mutation, FGFR1 mutation, EP300 mutation, MYC mutation, EZH2 mutation, JAK2 mutation, FBXW7 mutation, CCND3 mutation and GNA11 mutation , and where the method contains: (a) obtaining and/or receiving a first TIL population derived from a tumor resected from the individual by processing a tumor sample obtained from the individual into a plurality of tumor fragments; (b) adding the first TIL population to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is in an enclosure providing a first gas permeable surface area carried out in a container, wherein the first amplification is performed for about 3-14 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is performed without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion increasing for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the The transition from step (c) to step (d) is performed without opening the system; (e) collecting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system; and (f) transferring the collected TIL population from step (e) to an infusion bag, wherein the transfer from steps (e) to (f) is performed without opening the system; (g) cryopreserving the infusion bag comprising the collected TIL population from step (f) using a cryopreservation process; and (h) administering to the individual or patient a therapeutically effective dose of the third population of TILs from the infusion bag in step (g).

In some embodiments, the present invention provides a method of treating non-small cell lung cancer (NSCLC) by administering a population of tumor infiltrating lymphocytes (TIL) to a patient in need thereof, wherein the method comprises: (a) Test the PD-L1 expression of the patient's tumor and the tumor proportion score (TPS) of PD-L1, (b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from the group consisting of: EGFR mutation, EGFR insertion, EGFR exon 20 mutation, KRAS mutation, BRAF mutation, ALK mutation, c- ROS mutation (ROS1 mutation), ROS1 fusion, RET mutation, RET fusion, ERBB2 mutation, ERBB2 amplification, BRCA mutation, MAP2K1 mutation, PIK3CA, CDKN2A, PTEN mutation, UMD mutation, NRAS mutation, KRAS mutation, NF1 mutation, MET mutation , MET splicing and/or altered MET signaling, TP53 mutation, CREBBP mutation, KMT2C mutation, KMT2D mutation, ARID1A mutation, RB1 mutation, ATM mutation, SETD2 mutation, FLT3 mutation, PTPN11 mutation, FGFR1 mutation, EP300 mutation, MYC mutation, EZH2 mutation, JAK2 mutation, FBXW7 mutation, CCND3 mutation and GNA11 mutation, (c) determining that the patient has a PD-L1 TPS score of about 1% to about 49% and determining that the patient also has no driver mutations, (d) obtaining and/or receiving a first TIL population derived from a tumor resected from the individual by processing a tumor sample obtained from the individual into tumor fragments; (e) adding the first TIL population to the closed system; (f) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is in an enclosure providing a first gas permeable surface area in a container, wherein the first amplification is performed for about 3-14 days to obtain the second TIL population, and wherein the transition from step (e) to step (f) is performed without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion increasing for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the The transition from step (f) to step (g) is performed without opening the system; (h) collecting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system; and (i) transferring the collected TIL population from step (e) to an infusion bag, wherein the transfer from steps (e) to (f) is performed without opening the system; (j) cryopreserving the infusion bag comprising the collected TIL population from step (f) using a cryopreservation process; and (k) administering to the individual or patient a therapeutically effective dose of the third population of TILs from the infusion bag in step (g).

In some embodiments, the present invention provides a method of treating non-small cell lung cancer (NSCLC) by administering a population of tumor infiltrating lymphocytes (TIL) to a patient in need thereof, wherein the method comprises: (a) Test the PD-L1 expression of the patient's tumor and the tumor proportion score (TPS) of PD-L1, (b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from the group consisting of: EGFR mutation, EGFR insertion, EGFR exon 20 mutation, KRAS mutation, BRAF mutation, ALK mutation, c- ROS mutation (ROS1 mutation), ROS1 fusion, RET mutation, RET fusion, ERBB2 mutation, ERBB2 amplification, BRCA mutation, MAP2K1 mutation, PIK3CA, CDKN2A, PTEN mutation, UMD mutation, NRAS mutation, KRAS mutation, NF1 mutation, MET mutation , MET splicing and/or altered MET signaling, TP53 mutation, CREBBP mutation, KMT2C mutation, KMT2D mutation, ARID1A mutation, RB1 mutation, ATM mutation, SETD2 mutation, FLT3 mutation, PTPN11 mutation, FGFR1 mutation, EP300 mutation, MYC mutation, EZH2 mutation, JAK2 mutation, FBXW7 mutation, CCND3 mutation and GNA11 mutation, (c) determining that the patient has a TPS score of less than about 1% for PD-L1 and determining that the patient also has no driver mutations, (d) obtaining and/or receiving a first TIL population derived from a tumor resected from the individual by processing a tumor sample obtained from the individual into tumor fragments; (e) adding the first TIL population to the closed system; (f) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is in an enclosure providing a first gas permeable surface area in a container, wherein the first amplification is performed for about 3-14 days to obtain the second TIL population, and wherein the transition from step (e) to step (f) is performed without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion increasing for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the The transition from step (f) to step (g) is performed without opening the system; (h) collecting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system; and (i) transferring the collected TIL population from step (e) to an infusion bag, wherein the transfer from steps (e) to (f) is performed without opening the system; (j) cryopreserving the infusion bag comprising the collected TIL population from step (f) using a cryopreservation process; and (k) administering to the individual or patient a therapeutically effective dose of the third population of TILs from the infusion bag in step (g).

In some embodiments, the present invention provides a method of treating non-small cell lung cancer (NSCLC) by administering a population of tumor infiltrating lymphocytes (TIL) to a patient in need thereof, wherein the method comprises: (a) Test the PD-L1 expression of the patient's tumor and the tumor proportion score (TPS) of PD-L1, (b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from the group consisting of: EGFR mutation, EGFR insertion, KRAS mutation, BRAF mutation, ALK mutation, c-ROS mutation (ROS1 mutation) , ROS1 fusion, RET mutation or RET fusion, (c) determining that the patient has a PD-L1 TPS score of about 1% to about 49% and determining that the patient also has no driver mutations, (d) obtaining and/or receiving a first TIL population derived from a tumor resected from the individual by processing a tumor sample obtained from the individual into tumor fragments; (e) adding the first TIL population to the closed system; (f) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is in an enclosure providing a first gas permeable surface area in a container, wherein the first amplification is performed for about 3-14 days to obtain the second TIL population, and wherein the transition from step (e) to step (f) is performed without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion increasing for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the The transition from step (f) to step (g) is performed without opening the system; (h) collecting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system; and (i) transferring the collected TIL population from step (e) to an infusion bag, wherein the transfer from steps (e) to (f) is performed without opening the system; (j) cryopreserving the infusion bag comprising the collected TIL population from step (f) using a cryopreservation process; and (k) administering to the individual or patient a therapeutically effective dose of the third population of TILs from the infusion bag in step (g).

In some embodiments, the present invention provides a method of treating non-small cell lung cancer (NSCLC) by administering a population of tumor infiltrating lymphocytes (TIL) to a patient in need thereof, wherein the method comprises: (a) Test the PD-L1 expression of the patient's tumor and the tumor proportion score (TPS) of PD-L1, (b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from the group consisting of: EGFR mutation, EGFR insertion, KRAS mutation, BRAF mutation, ALK mutation, c-ROS mutation (ROS1 mutation) , ROS1 fusion, RET mutation or RET fusion, (c) determining that the patient has a TPS score of less than about 1% for PD-L1 and determining that the patient also has no driver mutations, (d) obtaining and/or receiving a first TIL population derived from a tumor resected from the individual by processing a tumor sample obtained from the individual into tumor fragments; (e) adding the first TIL population to the closed system; (f) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is in an enclosure providing a first gas permeable surface area in a container, wherein the first amplification is performed for about 3-14 days to obtain the second TIL population, and wherein the transition from step (e) to step (f) is performed without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion increasing for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the The transition from step (f) to step (g) is performed without opening the system; (h) collecting the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system; and (i) transferring the collected TIL population from step (e) to an infusion bag, wherein the transfer from steps (e) to (f) is performed without opening the system; (j) cryopreserving the infusion bag comprising the collected TIL population from step (f) using a cryopreservation process; and (k) administering to the individual or patient a therapeutically effective dose of the third population of TILs from the infusion bag in step (g).

In other embodiments, the invention provides a method for treating an individual with cancer comprising administering to the individual a therapeutically effective dose of a therapeutic TIL population described herein.

In other embodiments, the invention provides a method for treating an individual with cancer comprising administering to the individual a therapeutically effective dose of a TIL composition described herein.

In other embodiments, the present invention provides modified methods for treating an individual with a cancer described herein wherein a therapeutically effective dose of a therapeutic TIL population and TIL composition described herein is administered separately Previously, the subject had been administered a non-myeloablative lymphocyte-depleting regimen.

In other embodiments, the invention provides the methods described herein for treating an individual with cancer modified such that the non-myeloablative lymphocyte depletion regimen comprises the step of administering 60 mg/m per day ² doses of cyclophosphamide for two days, followed by daily doses of 25 mg/ ^m2 of fludarabine for five days.

In other embodiments, the present invention provides modified methods for treating an individual with a cancer described herein, the method further comprising treating the individual with a high-dose IL-2 regimen commencing on the day following administration of the TIL cells to the individual A step of.

In other embodiments, the present invention provides the methods described herein for treating an individual with cancer modified such that the high dose IL-2 regimen comprises administration as a 15 minute bolus intravenous infusion every eight hours 600,000 or 720,000 IU/kg until tolerated.

In other embodiments, the present invention provides methods described herein for treating an individual with cancer modified such that the cancer is a solid tumor.

In other embodiments, the present invention provides methods described herein for treating an individual with cancer modified such that the cancer is melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC) , lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancers caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, Kidney cancer or renal cell carcinoma.

In other embodiments, the invention provides methods modified for treating an individual with a cancer described herein, wherein the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM ) and gastrointestinal cancer.

In other embodiments, the present invention provides modified methods for treating an individual with a cancer described herein, wherein the cancer is melanoma.

In other embodiments, the present invention provides modified methods for treating an individual with a cancer described herein, wherein the cancer is HNSCC.

In other embodiments, the present invention provides modified methods for treating an individual with a cancer described herein, wherein the cancer is cervical cancer.

In other embodiments, the present invention provides modified methods for treating an individual with a cancer described herein, wherein the cancer is NSCLC.

In other embodiments, the present invention provides modified methods for treating an individual with a cancer described herein, wherein the cancer is glioblastoma (including GBM).

In other embodiments, the present invention provides modified methods for treating an individual with a cancer described herein, wherein the cancer is gastrointestinal cancer.

In other embodiments, the present invention provides modified methods for treating an individual with a cancer described herein, wherein the cancer is a hypermutated cancer.

In other embodiments, the present invention provides modified methods for treating an individual with a cancer described herein, wherein the cancer is a pediatric hypermutated cancer.

In other embodiments, the invention provides a therapeutic TIL population described herein for use in a method of treating an individual having cancer, the method comprising administering to the individual a therapeutically effective dose of the therapeutic TIL population.

In other embodiments, the invention provides a TIL composition described herein for use in a method of treating an individual having cancer, the method comprising administering to the individual a therapeutically effective amount of the TIL composition.

In other embodiments, the invention provides a therapeutic TIL population or TIL composition described herein modified such that upon administration of a therapeutically effective dose to the individual Prior to the described therapeutic TIL populations or the TIL compositions described herein, the subject has been administered a non-myeloablative lymphodepleting regimen.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the non-myeloablative lymphocyte depletion regimen comprises the step of: at a dose of 60 mg/m ² /day Cyclophosphamide was administered for two days, followed by fludarabine at a dose of 25 mg/ ^m2 /day for five days.

In other embodiments, the invention provides modified therapeutic TIL populations or TIL compositions described herein, further comprising the step of treating the patient with a high-dose IL-2 regimen starting on the day following administration of the TIL cells to the patient .

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the high-dose IL-2 regimen comprises 600,000 or 720,000 IL-2 administered as a 15-minute bolus intravenous infusion every eight hours. IU/kg until tolerated.

In other embodiments, the invention provides a therapeutic TIL population or TIL composition described herein that has been modified such that the cancer is a solid tumor.

In other embodiments, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the cancer is melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer Cancer, breast cancer, triple-negative breast cancer, cancers caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer or kidney cancer cell carcinoma.

In other embodiments, the invention provides modified therapeutic TIL populations or TIL compositions described herein, wherein the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and Gastrointestinal cancer.

In other embodiments, the invention provides a therapeutic TIL population or TIL composition described herein modified such that the cancer is melanoma.

In other embodiments, the invention provides a therapeutic TIL population or TIL composition described herein modified such that the cancer is HNSCC.

In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein modified such that the cancer is cervical cancer.

In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein modified such that the cancer is NSCLC.

In other embodiments, the invention provides a therapeutic TIL population or TIL composition described herein modified such that the cancer is glioblastoma.

In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein modified such that the cancer is gastrointestinal cancer.

In other embodiments, the invention provides a therapeutic TIL population or TIL composition described herein that has been modified such that the cancer is a hypermutated cancer.

In other embodiments, the invention provides a therapeutic TIL population or TIL composition described herein that has been modified such that the cancer is a pediatric hypermutational cancer.

In other embodiments, the invention provides the use of a therapeutic TIL population described herein in a method of treating cancer in an individual comprising administering to the individual a therapeutically effective dose of the therapeutic TIL population.

In other embodiments, the invention provides the use of the composition described in any of the preceding paragraphs in a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a TIL composition.

In other embodiments, the invention provides the use of a therapeutic TIL population as described herein or a TIL composition as described herein in a method of treating cancer in a patient comprising administering to the patient a non-myeloablative lymphocyte depletion Reduce the regimen, and then administer to the individual a therapeutically effective dose of any of the therapeutic TIL populations described in the preceding paragraphs or a therapeutically effective dose of the TIL composition described herein. B. Combination with PD-1 and PD-L1 inhibitors

In some embodiments, TIL therapy provided to cancer patients may comprise treatment with a therapeutic TIL population alone, or may comprise combination therapy comprising TILs and one or more PD-1 and/or PD-L1 inhibitors.

Planned death 1 (PD-1) is a 288 amino acid transmembrane immune checkpoint receptor protein expressed by T cells, B cells, natural killer (NK) T cells, activated monocytes and dendritic cells. PD-1, also known as CD279, belongs to the CD28 family and is encoded in humans by the Pdcd1 gene on chromosome 2. PD-1 consists of an immunoglobulin (Ig) superfamily domain, a transmembrane region, and an intracellular domain containing an immunoreceptor tyrosine inhibitory motif (ITIM) and an immunoreceptor tyrosine switching motif. body (ITSM). It is known that PD-1 and its ligands (PD-L1 and PD-L2) play an important role in immune tolerance, such as Keir et al., " Annu. Rev. Immunol. " 2008 , 26 , 677-704 as described. PD-1 provides inhibitory signals that negatively regulate T cell immune responses. PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273) are expressed on tumor cells and stromal cells, which may encounter activated T cells expressing PD-1, resulting in Immunosuppression of T cells. PD-L1 is a 290 amino acid transmembrane protein encoded by the Cd274 gene on human chromosome 9. Using PD-1 inhibitors, PD-L1 inhibitors and/or PD-L2 inhibitors to block the interaction between PD-1 and its ligands PD-L1 and PD-L2 can overcome immune resistance, as shown in recent clinical trials. Studies such as those described in Topalian et al., N. Eng. J. Med. 2012 , 366 , 2443-54 demonstrate. PD-L1 is expressed on many tumor cell lines, while PD-L2 is expressed mainly on dendritic cells and some tumor lines. In addition to T cells, which inducibly express PD-1 upon activation, PD-1 is also expressed on B cells, natural killer cells, macrophages, activated monocytes and dendritic cells.

In some embodiments, TILs and PD-1 inhibitors are administered as combination therapy or adjuvant therapy for the treatment of NSCLC.

In some embodiments, the NSCLC has not undergone prior therapy. In some embodiments, a PD-1 inhibitor is administered as first-line therapy or initial therapy. In some embodiments, a PD-1 inhibitor is administered as first-line therapy or initial therapy in combination with a TIL as described herein.

In some embodiments, the PD-1 inhibitor can be any PD-1 inhibitor or PD-1 blocker known in the art. In particular, it is one of the PD-1 inhibitors or blockers described in more detail in the following paragraphs. With respect to PD-1 inhibitors, the terms "inhibitor", "antagonist" and "blocker" are used interchangeably herein. For the avoidance of doubt, reference herein to a PD-1 inhibitor as an antibody may refer to a compound or an antigen-binding fragment, variant, conjugate or biosimilar thereof. For the avoidance of doubt, when referring to a PD-1 inhibitor herein, it may also refer to a small molecule compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, co-crystal or prodrug thereof.

In some embodiments, the PD-1 inhibitor is an antibody (ie, an anti-PD-1 antibody), a fragment thereof, including a Fab fragment or a single chain variable fragment (scFv) thereof. In some embodiments, the PD-1 inhibitor is a polyclonal antibody. In some embodiments, the PD-1 inhibitor is a monoclonal antibody. In some embodiments, the PD-1 inhibitor competes for binding to PD-1, and/or binds to an epitope on PD-1. In some embodiments, the antibody competes for binding to PD-1, and/or binds to an epitope on PD-1.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that binds human PD-1 with a KD of about 100 pM or less, binds with a KD of about 90 pM or less human PD-1, binds human PD-1 with a KD of about 80 pM or less, binds human PD-1 with a KD of about 70 pM or less, binds human PD-1 with a KD of about 60 pM or less, Binds human PD-1 with a KD of about 50 pM or less, binds human PD-1 with a KD of about 40 pM or less, binds human PD-1 with a KD of about 30 pM or less, binds human PD-1 with a KD of about 20 pM or less Binds human PD-1 with a lower KD, binds human PD-1 with a KD of about 10 pM or lower, or binds human PD-1 with a KD of about 1 pM or lower.

In some embodiments, the PD-1 inhibitor binds to human PD-1 with a k _assoc of about 7.5×10 ⁵ 1/M·s or faster, about 7.5×10 ⁵ 1/M·s or faster The k _assoc binds to human PD-1, the k assoc binds to human PD-1 at about 8×10 ⁵ 1/M·s or faster, the k _assoc binds to human PD-1 at about 8.5×10 ⁵ 1/M·s or faster _Assoc binds to human PD-1, binds to human PD-1 with a k _assoc of about 9×10 ⁵ 1/M·s or faster, binds with a k _assoc of about 9.5×10 ⁵ l/M·s or faster Inhibitors that bind to human PD-1, or bind to human PD-1 with a k _assoc of about 1×10 ⁶ 1/M·s or faster.

In some embodiments, the PD-1 inhibitor is _{a dissoc} that binds to human PD-1 with a k of about 2×10 ⁻⁵ 1/s or slower, a k of about 2.1×10 ⁻⁵ 1/s or slower _Dissoc binds to human PD-1, binds to human PD-1 with a k _dissoc of about 2.2×10 ^-5 1/s or slower, binds to human with a k _dissoc of about 2.3×10 ^-5 1/s or slower PD-1, binds to human PD-1 with a k _dissoc of about 2.4×10 ^-5 1/s or slower, binds to human PD-1 with a k _dissoc of about 2.5×10 ^-5 1/s or slower, Bind to human PD-1 with a k _dissoc of about 2.6×10 ^-5 1/s or slower, or bind to human PD-1 with a k _dissoc of about 2.7×10 ^-5 1/s or slower, with a k dissoc of about 2.8 Bind to human PD-1 with a k _dissoc of ×10 ^-5 1/s or slower, bind to human PD-1 with a k dissoc of about 2.9×10 ^-5 1/s or slower, or bind to human PD-1 with a k _dissoc of about 3×10 -5 1/ ^s or slower Inhibitor of ⁵ 1/s or slower k _dissoc binding to human PD-1.

In some embodiments, the PD-1 inhibitor is a PD-1 inhibitor that blocks or inhibits human PD-L1 or human PD-L2 and human PD with an IC50 of about 10 nM or less. -1 binding, blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 9 nM or lower, blocking or inhibiting human PD with an IC50 of about 8 nM or lower -L1 or human PD-L2 binding to human PD-1, blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 7 nM or less, at about 6 nM or Block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with a lower IC50, block or inhibit the binding of human PD-L1 or human PD-L2 to human PD with an IC50 of about 5 nM or lower -1 binding, blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower, blocking or inhibiting human PD with an IC50 of about 3 nM or lower -L1 or human PD-L2 binding to human PD-1, blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 2 nM or less, or at about 1 nM or lower IC50 to block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1.

In some embodiments, the PD-1 inhibitor is nivolumab (commercially available from Bristol-Myers Squibb as OPDIVO) or a biosimilar, antigen-binding fragment, conjugate or variant thereof. Nivolumab is a fully human IgG4 antibody that blocks the PD-1 receptor. In some embodiments, the anti-PD-1 antibody is an immunoglobulin G4κ anti-(human CD274) antibody. Nivolumab is assigned Chemical Abstracts Service (CAS) registry number 946414-94-4 and is also known as 5C4, BMS-936558, MDX-1106 and ONO-4538. The preparation and characterization of nivolumab is described in US Patent No. 8,008,449 and International Patent Publication No. WO 2006/121168, the disclosures of which are incorporated herein by reference. The clinical safety and efficacy of nivolumab in various forms of cancer have been described in Wang et al., Cancer Immunol Res. 2014, 2, 846-56; Page et al., Annual Med Review ( Ann.Rev. Med. ), 2014 , 65 , 185-202; and Weber et al., " J. Clin.Oncology ", 2013 , 31 , 4311-4318, these literatures The disclosure is incorporated herein by reference. The amino acid sequence of nivolumab is set forth in Table 18. Nivolumab at 22-96, 140-196, 254-314, 360-418, 22''-96'', 140''-196'', 254''-314'' and 360''-418 Intraheavy chain disulfide bonds at ''; Intralight chain disulfide bonds at 23'-88', 134'-194', 23'''-88''' and 134'''-194'''bond; heavy chain-light chain disulfide bond at 127-214', 127''-214'''; heavy chain-heavy chain disulfide bond at 219-219'' and 222-222''bond; and has N-glycosylation sites (H CH2 84.4) at 290, 290''.

In some embodiments, the PD-1 inhibitor comprises a heavy chain provided in SEQ ID NO: 158 and a light chain provided in SEQ ID NO: 159. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain respectively having the sequences shown in SEQ ID NO: 158 and SEQ ID NO: 159, or an antigen-binding fragment, a Fab fragment, a single chain variable Fragment (scFv), variant or conjugate. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain that are each at least 99% identical to the sequence set forth in SEQ ID NO: 158 and SEQ ID NO: 159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain that are each at least 98% identical to the sequence set forth in SEQ ID NO: 158 and SEQ ID NO: 159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain that are each at least 97% identical to the sequence set forth in SEQ ID NO: 158 and SEQ ID NO: 159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain that are each at least 96% identical to the sequence set forth in SEQ ID NO: 158 and SEQ ID NO: 159, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain that are each at least 95% identical to the sequence set forth in SEQ ID NO: 158 and SEQ ID NO: 159, respectively.

In some embodiments, the PD-1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of nivolumab. In some embodiments, the PD-1 inhibitor heavy chain variable region (V _H ) comprises the sequence set forth in SEQ ID NO: 160, and the PD-1 inhibitor light chain variable region (V _L ) comprises SEQ ID The sequence shown in NO:161 and its conservative amino acid substitutions. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region that are each at least 99% identical to the sequence set forth in SEQ ID NO: 160 and SEQ ID NO: 161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region that are each at least 98% identical to the sequence set forth in SEQ ID NO: 160 and SEQ ID NO: 161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region that are each at least 97% identical to the sequence set forth in SEQ ID NO: 160 and SEQ ID NO: 161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region that are each at least 96% identical to the sequence set forth in SEQ ID NO: 160 and SEQ ID NO: 161, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region that are each at least 95% identical to the sequence set forth in SEQ ID NO: 160 and SEQ ID NO: 161, respectively.

In some embodiments, the PD-1 inhibitor comprises heavy chain CDR1, CDR2 having the sequences set forth in SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 164, respectively, and conservative amino acid substitutions thereof and a CDR3 domain; and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 165, SEQ ID NO: 166 and SEQ ID NO: 167 and conservative amino acid substitutions thereof, respectively. In some embodiments, the antibody competes for binding to and/or binds to the same epitope on PD-1 as any of the foregoing antibodies.

In some embodiments, the PD-1 inhibitor is an anti-PD-1 biosimilar monoclonal antibody approved by drug regulatory agencies with reference to nivolumab. In some embodiments, the biosimilar comprises an anti-PD-1 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, An amino acid sequence with 99% or 100% sequence identity and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-1 antibody that has been authorized or applied for authorization, wherein the anti-PD-1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the The reference drug or reference biological product is nivolumab. Anti-PD-1 antibodies can be authorized by drug regulatory agencies, such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is nivolumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is nivolumab.

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered at a dose of about 200 mg, about 220 mg, about 240 mg, about 260 mg, About 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and nivolumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma and is administered at about 240 mg every 2 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma and is administered at about 480 mg every 4 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma, and about 1 mg/kg nivolumab is administered on the same day every 3 weeks, followed by 3 mg/kg i Pilimumab, 4 consecutive doses, followed by 240 mg every 2 weeks or 480 mg nivolumab every 4 weeks.

In some embodiments, nivolumab is administered for adjuvant treatment of melanoma. In some embodiments, nivolumab is administered at about 240 mg every 2 weeks for adjuvant treatment of melanoma. In some embodiments, nivolumab is administered at about 480 mg every 4 weeks for adjuvant treatment of melanoma. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered to treat metastatic non-small cell lung cancer. In some embodiments, nivolumab is administered at about 3 mg/kg every 2 weeks and ipilimumab is administered at about 1 mg/kg every 6 weeks to treat metastatic non-small cell lung cancer. In some embodiments, nivolumab is administered at about 360 mg every 3 weeks, plus 1 mg/kg ipilimumab every 6 weeks and 2 cycles of platinum-based doublet chemotherapy for the treatment of metastatic non-small cell lung cancer. In some embodiments, nivolumab is administered at about 240 mg every 2 weeks or 480 mg every 4 weeks for the treatment of metastatic non-small cell lung cancer. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered to treat small cell lung cancer. In some embodiments, nivolumab is administered at about 240 mg every 2 weeks to treat small cell lung cancer. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered at about 360 mg every 3 weeks and ipilimumab is administered at 1 mg/kg every 6 weeks to treat malignant pleural mesothelioma. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered to treat advanced renal cell carcinoma. In some embodiments, nivolumab is administered at about 240 mg every 2 weeks to treat advanced renal cell carcinoma. In some embodiments, nivolumab is administered at about 480 mg every 4 weeks to treat advanced renal cell carcinoma. In some embodiments, nivolumab is administered at about 3 mg/kg, followed by ipilimumab at about 1 mg/kg on the same day every 3 weeks for up to 4 doses, then every 2 weeks thereafter 240 mg nivolumab for advanced renal cell carcinoma. In some embodiments, nivolumab is administered at about 3 mg/kg, followed by ipilimumab at about 1 mg/kg on the same day every 3 weeks for up to 4 doses, then every 2 weeks thereafter 240 mg, 480 mg nivolumab every 4 weeks for the treatment of advanced renal cell carcinoma. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered to treat classical Hodgkin's lymphoma. In some embodiments, nivolumab is administered at about 240 mg every 2 weeks to treat classical Hodgkin's lymphoma. In some embodiments, nivolumab is administered at about 480 mg every 4 weeks to treat classical Hodgkin's lymphoma. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab is administered at about 240 mg every 2 weeks for the treatment of recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab is administered at about 480 mg every 4 weeks for the treatment of recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered at about 240 mg every 2 weeks for the treatment of locally advanced or metastatic urothelial carcinoma. In some embodiments, nivolumab is administered at about 480 mg every 4 weeks for the treatment of locally advanced or metastatic urothelial carcinoma. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered to treat microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, nivolumab is administered to treat adult and pediatric patients with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, nivolumab is administered at about 240 mg every 2 weeks to treat microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastasis in adult and pediatric patients > 40 kg colorectal cancer. In some embodiments, nivolumab is administered at about 480 mg every 4 weeks to treat microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastases in adult and pediatric patients > 40 kg colorectal cancer. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered at about 3 mg/kg every 2 weeks to treat microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastases in pediatric patients <40 kg colorectal cancer. In some embodiments, nivolumab is administered at about 3 mg/kg, followed by 1 mg/kg ipilimumab on the same day every 3 weeks for up to 4 doses, followed by 240 mg every 2 weeks Nivolumab for the treatment of microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients ≥40 kg. In some embodiments, nivolumab is administered at about 3 mg/kg, followed by 1 mg/kg ipilimumab every 3 weeks on the same day for 4 doses, followed by 480 mg every 4 weeks Nivolumab for the treatment of microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer in adult and pediatric patients ≥40 kg. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered to treat hepatocellular carcinoma. In some embodiments, nivolumab is administered at about 240 mg every 2 weeks to treat hepatocellular carcinoma. In some embodiments, nivolumab is administered at about 480 mg every 4 weeks to treat hepatocellular carcinoma. In some embodiments, nivolumab is administered at about 1 mg/kg, followed by 3 mg/kg ipilimumab on the same day every 3 weeks for up to 4 doses, followed by nivolumab every 2 weeks Monoclonal antibody 240 mg for the treatment of hepatocellular carcinoma. In some embodiments, nivolumab is administered at about 1 mg/kg, followed by 3 mg/kg ipilimumab on the same day every 3 weeks for 4 doses, followed by 480 mg every 4 weeks Nivolumab for the treatment of hepatocellular carcinoma. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, nivolumab is administered to treat squamous cell carcinoma of the esophagus. In some embodiments, nivolumab is administered at about 240 mg every 2 weeks to treat squamous cell carcinoma of the esophagus. In some embodiments, nivolumab is administered at about 480 mg every 4 weeks to treat squamous cell carcinoma of the esophagus. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, nivolumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, the PD-1 inhibitor comprises pembrolizumab (commercially available as KEYTRUDA from Merck & Co., Kenilworth, NJ, USA) or an antigen-binding fragment, conjugate or variant. Pembrolizumab has been assigned CAS Registry Number 1374853-91-4 and is also known as Lanlizumab, MK-3475 and SCH-900475. Pembrolizumab has an immunoglobulin G4 anti-(human protein PDCD1 (programmed cell death 1)) antibody that contains (human house mouse monoclonal heavy chain) disulfide bond dimerization with human house mouse monoclonal light chain body structure. The structure of pembrolizumab can also be described as an immunoglobulin G4 anti-(human programmed cell death 1) antibody; contains humanized mouse monoclonal [228-L-proline (H10-S>P)] γ4 heavy chain (134-218') disulfide bond and humanized mouse single strain κ light chain dimer (226-226'':229-229'') disulfide bond. The properties, uses and preparation of pembrolizumab are described in International Patent Publication No. WO 2008/156712 A1, U.S. Patent No. 8,354,509, and U.S. Patent Application Publication Nos. US 2010/0266617 A1, US 2013/0108651 A1 No. and No. US 2013/0109843 A2, the disclosures of these patents are incorporated herein by reference. The clinical safety and efficacy of pembrolizumab in various forms of cancer are described in Fuerst, Oncology Times, 2014, 36, 35-36; Robert et al., The Lancet , 2014, 384, 1109-17; and Thomas et al., "Exp. Opin. Biol. Ther.", 2014, 14, 1061-1064. The amino acid sequence of pembrolizumab is set forth in Table 19. Pembrolizumab includes the following disulfide bonds: 22-96, 22''-96'', 23'-92', 23'''-92''', 134-218', 134''-218' '', 138'-198', 138'''-198''', 147-203, 147''-203'', 226-226'', 229-229'', 261-321, 261'' -321'', 367-425 and 367''-425''; and the following glycosylation sites (N): Asn-297 and Asn-297''. Pembrolizumab is an IgG4/κ isotype containing a stabilizing S228P mutation in the Fc region; insertion of this mutation in the IgG4 hinge region prevents formation of the half-molecule normally observed with IgG4 antibodies. Pembrolizumab is heterogeneously glycosylated at Asn297 within the Fc domain of each heavy chain, resulting in a molecular weight of approximately 149 kDa for the intact antibody. The major glycoform of pembrolizumab is the fucosylated degalactosyl doublet glycan form (GOF).

In some embodiments, the PD-1 inhibitor comprises a heavy chain provided by SEQ ID NO: 168 and a light chain provided by SEQ ID NO: 169. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain respectively having the sequences shown in SEQ ID NO: 168 and SEQ ID NO: 169, or an antigen-binding fragment, a Fab fragment, a single chain variable Fragment (scFv), variant or conjugate. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain that are each at least 99% identical to the sequence set forth in SEQ ID NO: 168 and SEQ ID NO: 169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain that are each at least 98% identical to the sequence set forth in SEQ ID NO: 168 and SEQ ID NO: 169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain that are each at least 97% identical to the sequence set forth in SEQ ID NO: 168 and SEQ ID NO: 169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain that are each at least 96% identical to the sequence set forth in SEQ ID NO: 168 and SEQ ID NO: 169, respectively. In some embodiments, the PD-1 inhibitor comprises a heavy chain and a light chain that are each at least 95% identical to the sequence set forth in SEQ ID NO: 168 and SEQ ID NO: 169, respectively.

In some embodiments, the PD-1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of pembrolizumab. In some embodiments, the PD-1 inhibitor heavy chain variable region (V _H ) comprises the sequence set forth in SEQ ID NO: 170, and the PD-1 inhibitor light chain variable region (V _L ) comprises SEQ ID The sequence shown in NO:171, or conservative amino acid substitutions thereof. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region that are each at least 99% identical to the sequence set forth in SEQ ID NO: 170 and SEQ ID NO: 171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region that are each at least 98% identical to the sequence set forth in SEQ ID NO: 170 and SEQ ID NO: 171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region that are each at least 97% identical to the sequence set forth in SEQ ID NO: 170 and SEQ ID NO: 171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region that are each at least 96% identical to the sequence set forth in SEQ ID NO: 170 and SEQ ID NO: 171, respectively. In some embodiments, the PD-1 inhibitor comprises a _VH region and a _VL region that are each at least 95% identical to the sequence set forth in SEQ ID NO: 170 and SEQ ID NO: 171, respectively.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2 having the sequences shown in SEQ ID NO: 172, SEQ ID NO: 173, and SEQ ID NO: 174, or conservative amino acid substitutions thereof, respectively and a CDR3 domain; and light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 175, SEQ ID NO: 176 and SEQ ID NO: 177, or conservative amino acid substitutions thereof, respectively. In some embodiments, the antibody competes for binding to and/or binds to the same epitope on PD-1 as any of the foregoing antibodies.

In some embodiments, the PD-1 inhibitor is an anti-PD-1 biosimilar monoclonal antibody approved by the drug regulatory agency with reference to pembrolizumab. In some embodiments, the biosimilar comprises an anti-PD-1 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, An amino acid sequence with 99% or 100% sequence identity and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-1 antibody that has been authorized or applied for authorization, wherein the anti-PD-1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the The reference drug or reference biological product is pembrolizumab. Anti-PD-1 antibodies can be authorized by drug regulatory agencies, such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is pembrolizumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is pembrolizumab.

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and pembrolizumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and pembrolizumab is administered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or About 10 mg/kg. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and nivolumab is administered at a dose of about 200 mg, about 220 mg, about 240 mg, about 260 mg , about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks . In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered to treat melanoma. In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat melanoma. In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat melanoma. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered to treat NSCLC. In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat NSCLC. In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat NSCLC. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered to treat small cell lung cancer (SCLC). In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat SCLC. In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat SCLC. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered to treat head and neck squamous cell carcinoma (HNSCC). In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat HNSCC. In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat HNSCC. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat classical Hodgkin's lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks in an adult for the treatment of classical Hodgkin's lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some embodiments, the pediatric administration of pembrolizumab at about 2 mg/kg (up to 200 mg) every 3 weeks for the treatment of classical Hodgkin's lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL). In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat urothelial cancer. In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat urothelial carcinoma. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) cancer. In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks in an adult for the treatment of MSI-H or dMMR cancer. In some embodiments, the pediatric is administered pembrolizumab at about 2 mg/kg (up to 200 mg) every 3 weeks to treat MSI-H or dMMR cancer. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat microsatellite instability-high (MSI-H) or mismatch repair deficient colorectal cancer (dMMR CRC. In some embodiments , administering pembrolizumab at about 400 mg every 6 weeks for the treatment of MSI-H or dMMR CRC. In some embodiments, pembrolizumab is initiated 1, 2, 3, 4, or 5 days after IL-2 administration Pembrolizumab administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can also be administered prior to resection (i.e., at Pembrolizumab is administered 1, 2, 3, 4, or 5 weeks prior to obtaining a tumor sample from the individual or patient. In some embodiments, pembrolizumab may also be administered prior to resection (i.e., after the tumor sample is obtained from the individual or patient Pembrolizumab was administered 1, 2, or 3 weeks before).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat gastric cancer. In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat gastric cancer. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat esophageal cancer. In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat esophageal cancer. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat cervical cancer. In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat cervical cancer. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks for the treatment of hepatocellular carcinoma (HCC). In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat HCC. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to an adult for the treatment of Merkel cell carcinoma (MCC). In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat MCC in an adult. In some embodiments, the pediatric is administered pembrolizumab at about 2 mg/kg (up to 200 mg) every 3 weeks for the treatment of MCC. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks for the treatment of renal cell carcinoma (RCC). In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks and axitinib 5 mg is administered orally twice daily to treat RCC. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks to treat endometrial cancer. In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks and lenvatinib is administered orally once daily at 20 mg for non-MSI-H or dMMR tumors to treat endometrial cancer. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks in an adult for the treatment of a tumor mutational high burden (TMB-H) cancer. In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks in an adult for the treatment of TMB-H cancer. In some embodiments, the pediatric is administered pembrolizumab at about 2 mg/kg (up to 200 mg) every 3 weeks to treat TMB-H cancer. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks for the treatment of cutaneous squamous cell carcinoma (cSCC). In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat cSCC. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, pembrolizumab is administered at about 200 mg every 3 weeks for the treatment of triple negative breast cancer (TNBC). In some embodiments, pembrolizumab is administered at about 400 mg every 6 weeks to treat TNBC. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, if the patient or subject is an adult, ie, an adult indication is being treated, an additional dosing regimen of 400 mg every 6 weeks may be used. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, the PD-1 inhibitor is a commercially available anti-PD-1 monoclonal antibody, such as anti-m-PD-1 strain J43 (Cat. No. BE0033-2) and RMP1-14 (Cat. No. BE0146) (Bio X Cell, Inc., West Lebanon, NH, USA). A variety of commercially available anti-PD-1 antibodies are known to those of ordinary skill in the art.

In some embodiments, the PD-1 inhibitor is an antibody disclosed in U.S. Patent No. 8,354,509 or U.S. Patent Application Publication Nos. 2010/0266617 A1, 2013/0108651 A1, 2013/0109843 A2, which The disclosures of these patents are incorporated herein by reference. In some embodiments, the PD-1 inhibitor is described in U.S. Patent Nos. 8,287,856, 8,580,247, and 8,168,757, and U.S. Patent Application Publication Nos. 2009/0028857 A1, 2010/0285013 A1, 2013/ Anti-PD-1 antibodies in No. 0022600 A1 and No. 2011/0008369 A1, the teaching contents of these patents are incorporated herein by reference. In other embodiments, the PD-1 inhibitor is an anti-PD-1 antibody disclosed in US Pat. No. 8,735,553 B1, the disclosure of which is incorporated herein by reference. In some embodiments, the PD-1 inhibitor is pilizumab, also known as CT-011, which is described in US Patent No. 8,686,119, the disclosure of which is incorporated herein by reference.

二唑化合物及衍生物，諸如美國專利申請公開案第2015/0073024號中所描述之1,2,4-

二唑化合物及衍生物；環狀肽模擬化合物及衍生物，諸如美國專利申請公開案第US 2015/0073042號中所描述之環狀肽模擬化合物及衍生物；環狀化合物及衍生物，諸如美國專利申請公開案第US 2015/0125491中所描述之環狀化合物及衍生物；1,3,4-

二唑及1,3,4-噻二唑化合物及衍生物，諸如國際專利申請公開案第WO 2015/033301號中所描述之1,3,4-

二唑及1,3,4-噻二唑化合物及衍生物；基於肽之化合物及衍生物，諸如國際專利申請公開案第WO 2015/036927號及第WO 2015/04490號中所描述之基於肽之化合物及衍生物；或基於肽之巨環化合物及衍生物，諸如美國專利申請公開案第US 2014/0294898號中所描述之基於肽之巨環化合物及衍生物；該等專利中之每一者之揭示內容以全文引用之方式併入本文中。在一些實施例中，PD-1抑制劑係西普利單抗(cemiplimab)，其可購自Regeneron, Inc.。 In some embodiments, the PD-1 inhibitor can be a small molecule or a peptide or a peptide derivative, such as those disclosed in U.S. Patent Nos. 8,907,053, 9,096,642, and 9,044,442 and U.S. Patent Application Publication No. US 2015/0087581 Small molecules or peptides or peptide derivatives described; 1,2,4-

Oxadiazole compounds and derivatives, such as 1,2,4-

Oxadiazole compounds and derivatives; cyclic peptidomimetic compounds and derivatives such as those described in U.S. Patent Application Publication No. US 2015/0073042; cyclic compounds and derivatives such as those described in U.S. Patent Application Publication No. US 2015/0073042 Cyclic compounds and derivatives described in Patent Application Publication No. US 2015/0125491; 1,3,4-

Oxadiazole and 1,3,4-thiadiazole compounds and derivatives, such as the 1,3,4-thiadiazoles described in International Patent Application Publication No. WO 2015/033301

Oxadiazole and 1,3,4-thiadiazole compounds and derivatives; peptide-based compounds and derivatives, such as those described in International Patent Application Publication Nos. WO 2015/036927 and WO 2015/04490 or peptide-based macrocyclic compounds and derivatives, such as those described in U.S. Patent Application Publication No. US 2014/0294898; each of these patents The disclosure content of the author is incorporated herein by reference in its entirety. In some embodiments, the PD-1 inhibitor is cemiplimab, which is available from Regeneron, Inc.

In some embodiments, TILs and a PD-L1 inhibitor or PD-L2 inhibitor are administered as combination therapy or adjuvant therapy for the treatment of NSCLC.

In some embodiments, the NSCLC has not undergone prior therapy. In some embodiments, the PD-L1 inhibitor or PD-L2 inhibitor is administered as first-line therapy or initial therapy. In some embodiments, a PD-L1 inhibitor or PD-L2 inhibitor is administered as first-line therapy or initial therapy in combination with a TIL as described herein.

In some embodiments, the PD-L1 or PD-L2 inhibitor can be any PD-L1 or PD-L2 inhibitor, antagonist or blocker known in the art. In particular, it is one of the PD-L1 or PD-L2 inhibitors, antagonists or blockers described in more detail in the following paragraphs. With respect to PD-L1 and PD-L2 inhibitors, the terms "inhibitor", "antagonist" and "blocker" are used interchangeably herein. For the avoidance of doubt, reference herein to a PD-L1 or PD-L2 inhibitor as an antibody may refer to the compound or an antigen-binding fragment, variant, conjugate or biosimilar thereof. For the avoidance of doubt, when referring to a PD-L1 or PD-L2 inhibitor herein, it may also refer to a compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, co-crystal or prodrug thereof.

In some embodiments, the compositions, processes and methods described herein include a PD-L1 or PD-L2 inhibitor. In some embodiments, the PD-L1 or PD-L2 inhibitor is a small molecule. In some embodiments, the PD-L1 or PD-L2 inhibitor is an antibody (ie, an anti-PD-1 antibody), fragments thereof, including Fab fragments or single chain variable fragments (scFv) thereof. In some embodiments, the PD-L1 or PD-L2 inhibitor is a polyclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor is a monoclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor competes for binding to PD-L1 or PD-L2 and/or binds to an epitope on PD-L1 or PD-L2. In some embodiments, the antibody competes for binding to PD-L1 or PD-L2, and/or binds to an epitope on PD-L1 or PD-L2.

In some embodiments, the PD-L1 inhibitors provided herein are selective for PD-L1 in that the compound binds or interacts with PD-L1 at a concentration that it binds or interacts with other receptors, including the PD-L2 receptor. The concentration of interaction is much lower. In certain embodiments, the compound binds to the PD-L1 receptor with a binding constant that is at least about 2-fold higher, about 3-fold higher, About 5 times higher concentration, about 10 times higher concentration, about 20 times higher concentration, about 30 times higher concentration, about 50 times higher concentration, about 100 times higher concentration, about 200 times higher concentration, high About 300 times higher concentration or about 500 times higher concentration.

In some embodiments, the PD-L2 inhibitors provided herein are selective for PD-L2 in that the compound binds or interacts with PD-L2 at a concentration that it binds or interacts with other receptors, including the PD-L1 receptor. The concentration of interaction is much lower. In certain embodiments, the compound binds to the PD-L2 receptor with a binding constant that is at least about 2-fold higher, about 3-fold higher, About 5 times higher concentration, about 10 times higher concentration, about 20 times higher concentration, about 30 times higher concentration, about 50 times higher concentration, about 100 times higher concentration, about 200 times higher concentration, high About 300 times higher concentration or about 500 times higher concentration.

Without being bound by any theory, it is believed that tumor cells express PD-L1 and T cells express PD-1. However, the expression of PD-L1 by tumor cells is not required for the efficacy of PD-1 or PD-L1 inhibitors or blockers. In some embodiments, the tumor cells express PD-L1. In other embodiments, the tumor cells do not express PD-L1. In some embodiments, methods can include a combination of PD-1 and PD-L1 antibodies, such as the PD-1 and PD-L1 antibodies described herein, and TILs. Combinations of PD-1 and PD-L1 antibodies and TILs can be administered simultaneously or sequentially.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is a PD-L1 and/or PD-L2 inhibitor that is present at about 100 pM or less binding to human PD-L1 and/or PD-L2 with a KD of about 90 pM or less, binding to human PD-L1 and/or PD-L2 with a KD of about 80 pM or less, binding to human PD-L1 and / or PD-L2, bind human PD-L1 and/or PD-L2 with a KD of about 70 pM or less, bind human PD-L1 and/or PD-L2 with a KD of about 60 pM or less, bind human PD-L1 and/or PD-L2 with a KD of about 70 pM or less, Binds human PD-L1 and/or PD-L2 with a KD of 50 pM or less, binds human PD-L1 and/or PD-L2 with a KD of about 40 pM or less, or binds human PD-L1 and/or PD-L2 with a KD of about 30 pM or less Binds to human PD-L1 and/or PD-L2.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor binds to human PD-L1 and/or PD-L2 at a k _assoc of about 7.5×10 ⁵ 1/M·s or faster, and at about 8×10 ⁵ 1/M·s or faster k _assoc binds to human PD-L1 and/or PD-L2, and about 8.5×10 ⁵ 1/M·s or faster k _assoc binds to human PD- L1 and/or PD-L2, bind to human PD-L1 and/or PD-L2 with a k _assoc of about 9×10 ⁵ 1/M·s or faster, at about 9.5×10 ⁵ 1/M·s or Faster k _assoc binding to human PD-L1 and/or PD-L2, or inhibition of binding to human PD-L1 and/or PD-L2 with about 1×10 ⁶ 1/M·s or faster k _assoc agent.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor binds to human PD-L1 or PD-L2 at a k _dissoc of about 2×10 ⁻⁵ 1/s or slower, at about 2.1×10 ^-5 1/s or slower k _dissoc binding to human PD-1, about 2.2×10 ^-5 1/s or slower k _dissoc binding to human PD-1, about 2.3×10 ^-5 1/ s or slower k _dissoc binds to human PD-1, binds to human PD-1 with a k _dissoc of about 2.4×10 ^-5 1/s or slower, binds to human PD-1 at about 2.5×10 ^-5 1/s or slower Bind to human PD-1 with a k _dissoc of about 2.6×10 ^-5 1/s or slower, bind to human PD-1 with _{a k dissoc of} about 2.7×10 ^-5 1/s or slower Inhibitors that bind to human PD-L1 or PD-L2, or bind to human PD-L1 or PD-L2 with a k _dissoc of about 3×10 ^-5 1/s or slower.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is a PD-L1 and/or PD-L2 inhibitor, the PD-L1 and/or PD-L2 inhibitor is at about 10 nM or less Block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 9 nM or lower. Block or inhibit the binding of human PD-L1 or human PD-L2 to human PD-1 binding, blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 8 nM or lower, blocking or inhibiting human PD-L1 with an IC50 of about 7 nM or lower or the binding of human PD-L2 to human PD-1, blocking or inhibiting the binding of human PD-L1 or the binding of human PD-L2 to human PD-1 with an IC50 of about 6 nM or less, at about 5 nM or less Blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower, blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 binding, blocking or inhibiting the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower, blocking or inhibiting human PD-L1 with an IC50 of about 2 nM or lower Or the binding of human PD-L2 to human PD-1, or blocking human PD-1 with an IC50 of about 1 nM or lower or blocking the binding of human PD-L1 or human PD-L2 to human PD-1.

In some embodiments, the PD-L1 inhibitor is durvalumab, also known as MEDI4736 (commercially available from Medimmune, LLC, a subsidiary of AstraZeneca Pharmaceuticals, Inc., Gaithersburg, Maryland), or Antigen-binding fragments, conjugates or variants. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in US Patent No. 8,779,108 or US Patent Application Publication No. 2013/0034559, the disclosures of which are incorporated herein by reference. The clinical efficacy of durvalumab has been described in Page et al, Annual Medical Review, 2014, 65, 185-202; Brahmer et al, J Clinical Oncology 2014, 32, 5s (suppl, abstract 8021) and McDermott et al., Cancer Treatment Rev., 2014, 40, 1056-64. The preparation and characterization of durvalumab is described in US Patent No. 8,779,108, the disclosure of which is incorporated herein by reference. The amino acid sequence of durvalumab is set forth in Table 20. Durvalumab monoclonal antibodies include 22-96, 22''-96'', 23'-89', 23'''-89''', 135'-195', 135'''-195' '', 148-204, 148''-204'', 215'-224, 215''-224'', 230-230'', 233-233'', 265-325, 265''-325 '', disulfide bonds at 371-429 and 371''-429'; and N-glycosylation sites at Asn-301 and Asn-301''.

In some embodiments, the PD-L1 inhibitor comprises a heavy chain provided in SEQ ID NO: 178 and a light chain provided in SEQ ID NO: 179. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain respectively having the sequences shown in SEQ ID NO: 178 and SEQ ID NO: 179, or an antigen-binding fragment, a Fab fragment, a single chain variable Fragment (scFv), variant or conjugate. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 99% identical to the sequence set forth in SEQ ID NO: 178 and SEQ ID NO: 179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 98% identical to the sequence set forth in SEQ ID NO: 178 and SEQ ID NO: 179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 97% identical to the sequence set forth in SEQ ID NO: 178 and SEQ ID NO: 179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 96% identical to the sequence set forth in SEQ ID NO: 178 and SEQ ID NO: 179, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 95% identical to the sequence set forth in SEQ ID NO: 178 and SEQ ID NO: 179, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of durvalumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region (V _H ) comprises the sequence set forth in SEQ ID NO: 180, and the PD-L1 inhibitor light chain variable region (V _L ) comprises SEQ ID The sequence shown in NO:181, or conservative amino acid substitutions thereof. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 99% identical to the sequence set forth in SEQ ID NO: 180 and SEQ ID NO: 181, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 98% identical to the sequence set forth in SEQ ID NO: 180 and SEQ ID NO: 181, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 97% identical to the sequence set forth in SEQ ID NO: 180 and SEQ ID NO: 181, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 96% identical to the sequence set forth in SEQ ID NO: 180 and SEQ ID NO: 181, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 95% identical to the sequence set forth in SEQ ID NO: 180 and SEQ ID NO: 181, respectively.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2 having the sequences set forth in SEQ ID NO: 182, SEQ ID NO: 183, and SEQ ID NO: 184 and conservative amino acid substitutions thereof, respectively and a CDR3 domain; and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 185, SEQ ID NO: 186 and SEQ ID NO: 187 and conservative amino acid substitutions thereof, respectively. In some embodiments, the antibody competes for binding to and/or binds to the same epitope on PD-L1 as any of the foregoing antibodies.

In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 biosimilar monoclonal antibody approved by drug regulatory agencies with reference to durvalumab. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, An amino acid sequence with 99% or 100% sequence identity and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-L1 antibody that has been authorized or applied for authorization, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the The reference drug or reference biological product is durvalumab. Anti-PD-L1 antibodies can be authorized by drug regulatory agencies, such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is durvalumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is durvalumab.

In some embodiments, the PD-L1 inhibitor is avelumab, also known as MSB0010718C (commercially available from Merck/Serono), or an antigen-binding fragment, conjugate or variant thereof. The preparation and properties of avelumab are described in US Patent Application Publication No. US 2014/0341917 A1, the disclosure of which is specifically incorporated herein by reference. The amino acid sequence of avelumab is set forth in Table 21. Avelumab has 22-96, 147-203, 264-324, 370-428, 22''-96'', 147''-203'', 264''-324'' and 370''- Intraheavy chain disulfide bond at 428'' (C23-C104); 22'-90', 138'-197', 22'''-90''' and 138'''-197''' Disulfide bond in the light chain (C23-C104); heavy chain-light chain disulfide bond at 223-215' and 223''-215''' (h 5-CL 126); 229-229'' and Heavy chain at 232-232'' - intraheavy chain disulfide bond (h 11, h 14); N-glycosylation site at 300, 300'' (H CH2 N84.4); fucosyl and H CHS K2 C-terminal lysine clipping at 450 and 450'.

In some embodiments, the PD-L1 inhibitor comprises a heavy chain provided in SEQ ID NO: 188 and a light chain provided in SEQ ID NO: 189. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain respectively having the sequences shown in SEQ ID NO: 188 and SEQ ID NO: 189, or antigen-binding fragments, Fab fragments, single-chain variable chains thereof Fragment (scFv), variant or conjugate. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 99% identical to the sequence set forth in SEQ ID NO: 188 and SEQ ID NO: 189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 98% identical to the sequence set forth in SEQ ID NO: 188 and SEQ ID NO: 189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 97% identical to the sequence set forth in SEQ ID NO: 188 and SEQ ID NO: 189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 96% identical to the sequence set forth in SEQ ID NO: 188 and SEQ ID NO: 189, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 95% identical to the sequence set forth in SEQ ID NO: 188 and SEQ ID NO: 189, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of avelumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region (V _H ) comprises the sequence set forth in SEQ ID NO: 190, and the PD-L1 inhibitor light chain variable region (V _L ) comprises SEQ ID The sequence shown in NO:191, or conservative amino acid substitutions thereof. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 99% identical to the sequence set forth in SEQ ID NO: 190 and SEQ ID NO: 191, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 98% identical to the sequence set forth in SEQ ID NO: 190 and SEQ ID NO: 191, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 97% identical to the sequence set forth in SEQ ID NO: 190 and SEQ ID NO: 191, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 96% identical to the sequence set forth in SEQ ID NO: 190 and SEQ ID NO: 191, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 95% identical to the sequence set forth in SEQ ID NO: 190 and SEQ ID NO: 191, respectively.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2 having the sequences set forth in SEQ ID NO: 192, SEQ ID NO: 193, and SEQ ID NO: 194, respectively, and conservative amino acid substitutions thereof and a CDR3 domain; and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 195, SEQ ID NO: 196 and SEQ ID NO: 197 and conservative amino acid substitutions thereof, respectively. In some embodiments, the antibody competes for binding to and/or binds to the same epitope on PD-L1 as any of the foregoing antibodies.

In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 biosimilar monoclonal antibody approved by the drug regulatory agency with reference to avelumab. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, Amino acid sequences with 99% or 100% sequence identity, and which contain one or more post-translational modifications compared with the reference drug or reference biological product, wherein the reference drug or reference biological product is avelumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-L1 antibody that has been authorized or applied for authorization, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the The reference drug or reference biological product is avelumab. Anti-PD-L1 antibodies can be authorized by drug regulatory agencies, such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is avelumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is avelumab.

In some embodiments, the PD-L1 inhibitor is Atezolizumab, also known as MPDL3280A or RG7446 (commercially available as TECENTRIQ from Genentech, a subsidiary of Roche, Basel, Switzerland), or an antigen-binding fragment, conjugate thereof or variants. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in US Patent No. 8,217,149, the disclosure of which is specifically incorporated herein by reference. In some embodiments, the PD-L1 inhibitor is disclosed in US Patent Application Publication No. 2010/0203056 A1, No. 2013/0045200 A1, No. 2013/0045201 A1, No. 2013/0045202 A1 or No. 2014/ 0065135 A1, the disclosures of which patents are specifically incorporated herein by reference. The preparation and characterization of atezolizumab is described in US Patent No. 8,217,149, the disclosure of which is incorporated herein by reference. The amino acid sequence of atezolizumab is set forth in Table 22. Atezolizumab has 22-96, 145-201, 262-322, 368-426, 22''-96'', 145''-201'', 262''-322'' and 368'' Intraheavy chain disulfide bond at -426'' (C23-C104); 23'-88', 134'-194', 23'''-88''' and 134'''-194''' Intralight chain disulfide bond (C23-C104); 221-214' and 221''-214''' heavy chain-light chain disulfide bond (h 5-CL 126); 227-227'' And the heavy chain-intra-heavy chain disulfide bond at 230-230'' (h 11, h 14); and the N-glycosylation site at 298 and 298' (H CH2 N84.4>A).

In some embodiments, the PD-L1 inhibitor comprises a heavy chain provided in SEQ ID NO: 198 and a light chain provided in SEQ ID NO: 199. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 198 and SEQ ID NO: 199, respectively, or an antigen-binding fragment, a Fab fragment, a single chain variable Fragment (scFv), variant or conjugate. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 99% identical to the sequence set forth in SEQ ID NO: 198 and SEQ ID NO: 199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 98% identical to the sequence set forth in SEQ ID NO: 198 and SEQ ID NO: 199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 97% identical to the sequence set forth in SEQ ID NO: 198 and SEQ ID NO: 199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 96% identical to the sequence set forth in SEQ ID NO: 198 and SEQ ID NO: 199, respectively. In some embodiments, the PD-L1 inhibitor comprises a heavy chain and a light chain that are each at least 95% identical to the sequence set forth in SEQ ID NO: 198 and SEQ ID NO: 199, respectively.

In some embodiments, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of atezolizumab. In some embodiments, the PD-L1 inhibitor heavy chain variable region (V _H ) comprises the sequence set forth in SEQ ID NO: 200, and the PD-L1 inhibitor light chain variable region (V _L ) comprises SEQ ID The sequence shown in NO:201, or conservative amino acid substitutions thereof. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 99% identical to the sequence set forth in SEQ ID NO: 200 and SEQ ID NO: 201, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 98% identical to the sequence set forth in SEQ ID NO: 200 and SEQ ID NO: 201, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 97% identical to the sequence set forth in SEQ ID NO: 200 and SEQ ID NO: 201, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 96% identical to the sequence set forth in SEQ ID NO: 200 and SEQ ID NO: 201, respectively. In some embodiments, the PD-L1 inhibitor comprises a _VH region and a _VL region that are each at least 95% identical to the sequence set forth in SEQ ID NO: 200 and SEQ ID NO: 201, respectively.

In some embodiments, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2 having the sequences shown in SEQ ID NO: 202, SEQ ID NO: 203, and SEQ ID NO: 204, respectively, or conservative amino acid substitutions thereof and a CDR3 domain; and light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO:205, SEQ ID NO:206 and SEQ ID NO:207, or conservative amino acid substitutions thereof, respectively. In some embodiments, the antibody competes for binding to and/or binds to the same epitope on PD-L1 as any of the foregoing antibodies.

In some embodiments, the anti-PD-L1 antibody is an anti-PD-L1 biosimilar monoclonal antibody approved by drug regulatory agencies with reference to atezolizumab. In some embodiments, the biosimilar comprises an anti-PD-L1 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, Amino acid sequences with 99% or 100% sequence identity and which contain one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is atezolizumab . In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-L1 antibody that has been authorized or applied for authorization, wherein the anti-PD-L1 antibody is provided in a formulation different from that of the reference drug or reference biological product, wherein the The reference drug or reference biological product is atezolizumab. Anti-PD-L1 antibodies can be authorized by drug regulatory agencies, such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is atezolizumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is atezolizumab.

In some embodiments, PD-L1 inhibitors include those antibodies described in US Patent Application Publication No. US 2014/0341917 A1, the disclosure of which is incorporated herein by reference. In other embodiments, antibodies that compete with any of these antibodies for binding to PD-L1 are also included. In some embodiments, the anti-PD-L1 antibody is MDX-1105, also known as BMS-935559, which is disclosed in US Patent No. 7,943,743, the disclosure of which is incorporated herein by reference. In some embodiments, the anti-PD-L1 antibody is selected from the anti-PD-L1 antibodies disclosed in US Pat. No. 7,943,743, which is incorporated herein by reference.

In some embodiments, the PD-L1 inhibitor is a commercially available monoclonal antibody, such as INVIVOMAB anti-m-PD-L1 strain 10F.9G2 (Catalogue # BE0101, Bio X Cell, West Lebanon, NH, USA , Inc.). In some embodiments, the anti-PD-L1 antibody is a commercially available monoclonal antibody, such as AFFYMETRIX EBIOSCIENCE (MIH1). A variety of commercially available anti-PD-L1 antibodies are known to those of ordinary skill in the art.

In some embodiments, the PD-L2 inhibitor is a commercially available monoclonal antibody, such as BIOLEGEND 24F.10C12 Mouse IgG2aκ Isotype (Cat. No. 329602, Biolegend, Inc., San Diego, CA), SIGMA Anti-PD-L2 Antibody (Cat. No. SAB3500395, Sigma Alrich, St. Louis, MO) or other commercially available anti-PD-L2 antibodies known to those of ordinary skill in the art. C. Combinations with CTLA-4 inhibitors

In some embodiments, TIL therapy provided to a cancer patient may comprise treatment with a therapeutic TIL population alone, or may comprise combination therapy comprising TIL and one or more CTLA-4 inhibitors.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a member of the immunoglobulin superfamily and is expressed on the surface of helper T cells. CTLA-4 is a negative regulator of CD28-dependent T cell activation and serves as a checkpoint for the adaptive immune response. Similar to the T cell co-stimulatory protein CD28, CTLA-4 binds antigens to present CD80 and CD86 on cells. CTLA-4 delivers an inhibitory signal to T cells, while CD28 delivers a stimulatory signal. Human antibodies against human CTLA-4 have been described as immunostimulatory modulators of many disease states, such as treating or preventing viral and bacterial infections and treating cancer (WO 01/14424 and WO 00/37504). Several fully human anti-human CTLA-4 monoclonal antibodies (mAbs) have been studied in clinical trials for the treatment of various types of solid tumors, such antibodies include but are not limited to ipilimumab (MDX-010) and Qumei Monoclonal antibody (CP-675,206).

In some embodiments, the CTLA-4 inhibitor can be any CTLA-4 inhibitor or CTLA-4 blocker known in the art. In particular, it is one of the CTLA-4 inhibitors or blockers described in more detail in the following paragraphs. With respect to CTLA-4 inhibitors, the terms "inhibitor", "antagonist" and "blocker" are used interchangeably herein. For the avoidance of doubt, reference herein to a CTLA-4 inhibitor as an antibody may refer to the compound or an antigen-binding fragment, variant, conjugate or biosimilar thereof. For the avoidance of doubt, when referring to a CTLA-4 inhibitor herein, it may also refer to a small molecular compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, co-crystal or prodrug thereof.

CTLA-4 inhibitors suitable for use in the methods of the invention include, but are not limited to, anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA -4 antibody, monoclonal anti-CTLA-4 antibody, polyclonal anti-CTLA-4 antibody, chimeric anti-CTLA-4 antibody, MDX-010 (ipilimumab), tremezumab, anti-CD28 antibody, anti-CTLA -4 Adnectin, anti-CTLA-4 domain antibody, single-chain anti-CTLA-4 fragment, heavy chain anti-CTLA-4 fragment, light chain anti-CTLA-4 fragment, CTLA-4 inhibitor that promotes co-stimulatory pathway, Antibodies disclosed in PCT Publication No. WO 2001/014424, antibodies disclosed in PCT Publication No. WO 2004/035607, antibodies disclosed in US Publication No. 2005/0201994 and in the granted European Patent Antibodies in EP 1212422 B1, the disclosures of each of these patents are incorporated herein by reference. Additional CTLA-4 antibodies are described in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; PCT Publication Nos. WO 01/14424 and WO 00/37504; and U.S. Publication No. 2002/0039581 and 2002/086014, the disclosures of each of these patents are incorporated herein by reference. Other anti-CTLA-4 antibodies useful in the methods of the invention include, for example, those disclosed in WO 98/42752; U.S. Patent Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proceedings of the National Academy of Sciences, 95 (17):10067-10071 (1998); Camacho et al., Journal of Clinical Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Research, 58 :5301-5304 (1998); and US Patent Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281, the disclosures of each of which are incorporated herein by reference.

Additional CTLA-4 inhibitors include, but are not limited to, the following: capable of disrupting the ability of CD28 antigen to bind to its cognate ligand, usually by being activated, inhibiting the ability of CTLA-4 to bind to its cognate ligand, enhancing T cell response of the pathway, disrupting the ability of B7 to bind to CD28 and/or CTLA-4, disrupting the ability of B7 to activate co-stimulatory pathways, disrupting the ability of CD80 to bind to CD28 and/or CTLA-4, disrupting the ability of CD80 to activate co-stimulatory pathways ability, the ability to disrupt CD86 binding to CD28 and/or CTLA-4, the ability to disrupt CD86 to activate co-stimulatory pathways, and any inhibitor that disrupts co-stimulatory pathways. This must include: small molecule inhibitors of CD28, CD80, CD86, CTLA-4, and other members of the costimulatory pathway; antibodies against CD28, CD80, CD86, CTLA-4, and other members of the costimulatory pathway; antibodies against CD28, CD80 , CD86, CTLA-4, and other members of the costimulatory pathway; Adnectins against CD28, CD80, CD86, CTLA-4, and other members of the costimulatory pathway; CD28, CD80, CD86, CTLA-4 and RNAi inhibitors (single- and double-stranded) of other members of the costimulatory pathway; and other CTLA-4 inhibitors.

In some embodiments, the CTLA-4 inhibitor binds to CTLA-4 with a Kd of about 10 ⁻⁶ M or less, 10 ⁻⁷ M or less, 10 ⁻⁸ M or less, 10 ^{− 9} M or less, 10 ⁻¹⁰ M or less, 10 ⁻¹¹ M or less, 10 ⁻¹² M or less, for example between 10 ⁻¹³ M and 10 ⁻¹⁶ M, or between any two of the preceding values as Any range of endpoints. In some embodiments, the CTLA-4 inhibitor binds to CTLA-4 with a Kd no greater than 10 times the Kd of ipilimumab when compared using the same assay. In some embodiments, the CTLA-4 inhibitor binds to CTLA-4 with a Kd that is about the same as or less (e.g., up to 10-fold lower or up to 100-fold lower) than the Kd of ipilimumab when compared using the same assay . In some embodiments, the CTLA-4 inhibitor inhibits CTLA-4 and CD80 as compared to the IC50 value for ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively, when compared using the same assay. Or CD86 binding IC50 value is not more than 10 times higher. In some embodiments, the CTLA-4 inhibitor inhibits CTLA-4 and CD80 as compared to the IC50 value for ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively, when compared using the same assay. or CD86 binding with approximately the same or less (eg, up to 10-fold lower or up to 100-fold lower) IC50 values.

In some embodiments, the CTLA-4 inhibitor is used in an amount sufficient to inhibit the expression of CTLA-4 and/or reduce the biological activity of CTLA-4 by at least 20%, 30%, 40% relative to a suitable control %, 50%, 60%, 70%, 80%, 90%, 95% or 100%, for example between 50% and 75%, 75% and 90% or 90% and 100%. In some embodiments, the CTLA-4 pathway inhibitor is used in an amount sufficient to reduce binding of CTLA-4 to CD80, CD86, or both by at least 20%, 30%, 40% relative to a suitable control , 50%, 60%, 70%, 80%, 90%, 95% or 100%, such as a reduction between 50% and 75%, 75% and 90%, or 90% and 100% relative to a suitable control Biological activity of CTLA-4. A suitable control in the context of assessing or quantifying the effect of an agent of interest is typically a comparable biological system (e.g. a cell or an individual) that has not been exposed to or treated with the agent of interest (e.g. a CTLA-4 pathway inhibitor) (or have been exposed to or treated with negligible amounts). In some embodiments, the biological system can serve as its own control, eg, the biological system can be assessed prior to exposure to or treatment with the agent and compared to its state after the beginning or end of the exposure or treatment. In some embodiments, historical controls may be used.

In some embodiments, the CTLA-4 inhibitor is ipilimumab (commercially available as Yervoy from Bristol-Myers Squibb) or a biosimilar, antigen-binding fragment, conjugate or variant thereof. As known in the art, ipilimumab refers to the anti-CTLA-4 antibody, a fully human IgG1κ antibody derived from a transgenic mouse with human genes encoding heavy and light chains to generate a functional human lineage . Ipilimumab is also referred to by its CAS Registry Number 477202-00-9 and in PCT Publication No. WO 01/14424, which is incorporated by reference in its entirety for all purposes. It is disclosed in the form of antibody 10DI. In particular, ipilimumab comprises a light chain variable region and a heavy chain variable region (having a light chain variable region comprising SEQ ID NO:211 and having a heavy chain variable region comprising SEQ ID NO:210) . Pharmaceutical compositions of ipilimumab include all pharmaceutically acceptable compositions comprising ipilimumab and one or more diluents, vehicles or excipients. Examples of pharmaceutical compositions containing ipilimumab are described in International Patent Application Publication No. WO 2007/67959. Ipilimumab can be administered intravenously (IV).

In some embodiments, the CTLA-4 inhibitor comprises a heavy chain provided by SEQ ID NO:208 and a light chain provided by SEQ ID NO:209. In some embodiments, the CTLA-4 inhibitor comprises heavy and light chains or antigen-binding fragments thereof, Fab fragments, single chain variable fragments having the sequences shown in SEQ ID NO: 208 and SEQ ID NO: 209, respectively (scFv), variant or combination. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 99% identical to the sequence set forth in SEQ ID NO: 208 and SEQ ID NO: 209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 98% identical to the sequence set forth in SEQ ID NO: 208 and SEQ ID NO: 209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 97% identical to the sequence set forth in SEQ ID NO: 208 and SEQ ID NO: 209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 96% identical to the sequence set forth in SEQ ID NO: 208 and SEQ ID NO: 209, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 95% identical to the sequence set forth in SEQ ID NO: 208 and SEQ ID NO: 209, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of ipilimumab. In some embodiments, the CTLA-4 inhibitor heavy chain variable region (V _H ) comprises the sequence set forth in SEQ ID NO: 210, and the CTLA-4 inhibitor light chain variable region (V _L ) comprises SEQ ID The sequence shown in NO:211, or conservative amino acid substitutions thereof. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 99% identical to the sequence set forth in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 98% identical to the sequence set forth in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 97% identical to the sequence set forth in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 96% identical to the sequence set forth in SEQ ID NO:210 and SEQ ID NO:211, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 95% identical to the sequence set forth in SEQ ID NO:210 and SEQ ID NO:211, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2 having the sequences shown in SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:214, respectively, or conservative amino acid substitutions thereof and a CDR3 domain; and light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO:215, SEQ ID NO:216 and SEQ ID NO:217, or conservative amino acid substitutions thereof, respectively. In some embodiments, the antibody competes for binding to and/or binds to the same epitope on CTLA-4 as any of the foregoing antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonal antibody approved by drug regulatory agency with reference to ipilimumab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, An amino acid sequence with 99% or 100% sequence identity and comprising one or more post-translational modifications compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of ipilimumab is set forth in Table 23. In some embodiments, the biosimilar is an anti-CTLA-4 antibody licensed or applying for authorization, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the The reference drug or reference biological product is ipilimumab. Anti-CTLA-4 antibodies can be authorized by drug regulatory agencies, such as US FDA and/or EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is ipilimumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is ipilimumab.

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or About 10 mg/kg. In some embodiments, ipilimumab administration may also begin 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, ipilimumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, wherein ipilimumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered at a dose of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, ipilimumab administration begins 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, ipilimumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, and ipilimumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks . In some embodiments, ipilimumab administration may also begin 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, ipilimumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, ipilimumab is administered to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab is administered at about mg/kg every 3 weeks for up to 4 doses to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab administration may also begin 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, ipilimumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, ipilimumab is administered for adjuvant treatment of melanoma. In some embodiments, ipilimumab is administered at about 10 mg/kg every 3 weeks for 4 doses, followed by 10 mg/kg every 12 weeks for up to 3 years, for adjuvant treatment of melanoma. In some embodiments, ipilimumab administration may also begin 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, ipilimumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, ipilimumab is administered to treat advanced renal cell carcinoma. In some embodiments, ipilimumab is administered at about 1 mg/kg every 3 weeks followed by 3 mg/kg nivolumab on the same day for 4 consecutive doses to treat advanced renal cell carcinoma . In some embodiments, following completion of the 4 doses of the combination, nivolumab may be administered as a single agent for advanced renal cell carcinoma and/or renal cell carcinoma according to standard dosing regimens. In some embodiments, ipilimumab administration may also begin 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, ipilimumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, ipilimumab is administered to treat microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, about 1 mg/kg of ipilimumab is administered intravenously over 30 minutes every 3 weeks, followed by 3 mg/kg of nivolumab administered intravenously over 30 minutes on the same day for 4 doses for the treatment of microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. In some embodiments, following completion of 4 doses of the composition, as recommended according to standard dosing regimens for microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer with Nivolumab was administered as a single agent. In some embodiments, ipilimumab administration may also begin 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, ipilimumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, ipilimumab is administered to treat hepatocellular carcinoma. In some embodiments, ipilimumab is administered intravenously at about 3 mg/kg over 30 minutes every 3 weeks, followed by nivolumab at 1 mg/kg intravenously over 30 minutes on the same day for 4 doses to treat hepatocellular carcinoma. In some embodiments, after completion of the 4 doses of the combination, nivolumab is administered as a single agent for hepatocellular carcinoma according to a standard dosing regimen. In some embodiments, ipilimumab administration may also begin 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, ipilimumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, ipilimumab is administered to treat metastatic non-small cell lung cancer. In some embodiments, ipilimumab is administered at about 1 mg/kg every 6 weeks and nivolumab is administered at 3 mg/kg every 2 weeks to treat metastatic non-small cell lung cancer. In some embodiments, ipilimumab is administered at about 1 mg/kg every 6 weeks, plus nivolumab 360 mg every 3 weeks and 2 cycles of platinum-based doublet chemotherapy for the treatment of metastatic non-small cell lung cancer. In some embodiments, ipilimumab administration may also begin 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, ipilimumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, ipilimumab is administered to treat malignant pleural mesothelioma. In some embodiments, ipilimumab is administered at about 1 mg/kg every 6 weeks and nivolumab 360 mg every 3 weeks to treat malignant pleural mesothelioma. In some embodiments, ipilimumab administration may also begin 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, ipilimumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

Tremezumab (also known as CP-675,206) is a fully human IgG2 monoclonal antibody with CAS number 745013-59-6. Tremezumab is disclosed as antibody 11.2.1 in US Patent No. 6,682,736 (herein incorporated by reference). The amino acid sequences of the heavy and light chains of Tremezumab are set forth in SEQ IND NO: 218 and 219, respectively. Tremezumab has been studied in clinical trials for the treatment of a variety of tumors including melanoma and breast cancer; administered intravenously in single or multiple doses every 4 or 12 weeks in a dose range between 0.01 and 15 mg/kg Administration of tremelimumab. In the regimens provided by the present invention, tremelimumab is administered topically, especially intradermally or subcutaneously. The effective amount of Tremezumab administered intradermally or subcutaneously is generally in the range of 5-200 mg/dose per subject. In some embodiments, the effective amount of Tremezumab is in the range of 10-150 mg/dose per person per dose. In some specific embodiments, the effective amount of Tremezumab is about 10, 25, 37.5, 40, 50, 75, 100, 125, 150, 175 or 200 mg/dose per person.

In some embodiments, the CTLA-4 inhibitor comprises a heavy chain provided by SEQ ID NO:218 and a light chain provided by SEQ ID NO:219. In some embodiments, the CTLA-4 inhibitor comprises heavy and light chains or antigen-binding fragments thereof, Fab fragments, single chain variable fragments having the sequences shown in SEQ ID NO: 218 and SEQ ID NO: 219, respectively (scFv), variant or combination. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 99% identical to the sequence set forth in SEQ ID NO: 218 and SEQ ID NO: 219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 98% identical to the sequence set forth in SEQ ID NO: 218 and SEQ ID NO: 219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 97% identical to the sequence set forth in SEQ ID NO: 218 and SEQ ID NO: 219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 96% identical to the sequence set forth in SEQ ID NO: 218 and SEQ ID NO: 219, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 95% identical to the sequence set forth in SEQ ID NO: 218 and SEQ ID NO: 219, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of Tremezumab. In some embodiments, the CTLA-4 inhibitor heavy chain variable region (V _H ) comprises the sequence set forth in SEQ ID NO: 220, and the CTLA-4 inhibitor light chain variable region (V _L ) comprises SEQ ID The sequence shown in NO:221, or conservative amino acid substitutions thereof. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 99% identical to the sequence set forth in SEQ ID NO: 220 and SEQ ID NO: 221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 98% identical to the sequence set forth in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 97% identical to the sequence set forth in SEQ ID NO: 220 and SEQ ID NO: 221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 96% identical to the sequence set forth in SEQ ID NO:220 and SEQ ID NO:221, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 95% identical to the sequence set forth in SEQ ID NO:220 and SEQ ID NO:221, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2 having the sequences set forth in SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224, respectively, and conservative amino acid substitutions thereof and a CDR3 domain; and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:225, SEQ ID NO:226 and SEQ ID NO:227 and conservative amino acid substitutions thereof, respectively. In some embodiments, the antibody competes for binding to and/or binds to the same epitope on CTLA-4 as any of the foregoing antibodies.

In some embodiments, the CTLA-4 inhibitor is an anti-CTLA-4 biosimilar monoclonal antibody approved by drug regulatory agencies with reference to tremelimumab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, An amino acid sequence with 99% or 100% sequence identity and comprising one or more post-translational modifications compared with the reference drug or reference biological product, wherein the reference drug or reference biological product is Tremezumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of Tremezumab is set forth in Table 24. In some embodiments, the biosimilar is an anti-CTLA-4 antibody licensed or applying for authorization, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the The reference drug or reference biological product is Tremezumab. Anti-CTLA-4 antibodies can be authorized by drug regulatory agencies, such as US FDA and/or EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is Tremezumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is Tremezumab.

In some embodiments, the CTLA-4 inhibitor is Tremezumab or a biosimilar thereof, and Tremezumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is Tremezumab or a biosimilar thereof, and Tremezumab is administered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, or about 10 mg/kg. In some embodiments, tremelimumab administration can also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, tremezumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, the CTLA-4 inhibitor is Tremezumab or a biosimilar thereof, wherein Tremezumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is Tremezumab or a biosimilar thereof, and Tremezumab is administered at a dose of about 200 mg, about 220 mg, about 240 mg, about 260 mg, About 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, administration of tremelimumab begins 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, tremezumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, the CTLA-4 inhibitor is Tremezumab or a biosimilar thereof, and Tremezumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, tremelimumab administration can also be initiated 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient). In some embodiments, tremezumab administration can also be initiated 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the individual or patient).

In some embodiments, the CTLA-4 inhibitor is Zefelizumab from Agenus or a biosimilar, antigen-binding fragment, conjugate or variant thereof. Zefelizumab is a fully human monoclonal antibody. Zefelizumab has been assigned Chemical Abstracts Service (CAS) registry number 2148321-69-9 and is also known as AGEN1884. The preparation and characterization of Zefelizumab is described in US Patent No. 10,144,779 and US Patent Application Publication No. US2020/0024350 A1, the disclosures of which are incorporated herein by reference.

In some embodiments, the CTLA-4 inhibitor comprises a heavy chain provided by SEQ ID NO:228 and a light chain provided by SEQ ID NO:229. In some embodiments, the CTLA-4 inhibitor comprises heavy and light chains or antigen-binding fragments thereof, Fab fragments, single chain variable fragments having the sequences shown in SEQ ID NO: 228 and SEQ ID NO: 229, respectively (scFv), variant or combination. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 99% identical to the sequence set forth in SEQ ID NO: 228 and SEQ ID NO: 229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 98% identical to the sequence set forth in SEQ ID NO: 228 and SEQ ID NO: 229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 97% identical to the sequence set forth in SEQ ID NO: 228 and SEQ ID NO: 229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 96% identical to the sequence set forth in SEQ ID NO: 228 and SEQ ID NO: 229, respectively. In some embodiments, the CTLA-4 inhibitor comprises a heavy chain and a light chain that are each at least 95% identical to the sequence set forth in SEQ ID NO: 228 and SEQ ID NO: 229, respectively.

In some embodiments, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of Zefelizumab. In some embodiments, the CTLA-4 inhibitor heavy chain variable region (V _H ) comprises the sequence set forth in SEQ ID NO: 230, and the CTLA-4 inhibitor light chain variable region (V _L ) comprises SEQ ID The sequence shown in NO:231, or conservative amino acid substitutions thereof. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 99% identical to the sequence set forth in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 98% identical to the sequence set forth in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 97% identical to the sequence set forth in SEQ ID NO:230 and SEQ ID NO:231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 96% identical to the sequence set forth in SEQ ID NO: 230 and SEQ ID NO: 231, respectively. In some embodiments, the CTLA-4 inhibitor comprises a _VH region and a _VL region that are each at least 95% identical to the sequence set forth in SEQ ID NO: 230 and SEQ ID NO: 231, respectively.

In some embodiments, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2 having the sequences shown in SEQ ID NO:231, SEQ ID NO:233, and SEQ ID NO:234, respectively, or conservative amino acid substitutions thereof and a CDR3 domain; and light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 235, SEQ ID NO: 236 and SEQ ID NO: 237, or conservative amino acid substitutions thereof, respectively. In some embodiments, the antibody competes for binding to and/or binds to the same epitope on CTLA-4 as any of the foregoing antibodies.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 biosimilar monoclonal antibody approved by drug regulatory agency with reference to Zefelizumab. In some embodiments, the biosimilar comprises an anti-CTLA-4 antibody comprising at least 97% sequence identity, e.g., 97%, 98%, Amino acid sequences with 99% or 100% sequence identity, and which contain one or more post-translational modifications compared with the reference drug or reference biological product, wherein the reference drug or reference biological product is Zefelizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequence of Zefelizumab is set forth in Table 25. In some embodiments, the biosimilar is an anti-CTLA-4 antibody licensed or applying for authorization, wherein the anti-CTLA-4 antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the The reference drug or reference biological product is Zefelizumab. Anti-CTLA-4 antibodies can be authorized by drug regulatory agencies, such as US FDA and/or EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is Zefelizumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, wherein the The reference drug or reference biological product is Zefelizumab.

Examples of additional anti-CTLA-4 antibodies include, but are not limited to: AGEN1181, BMS-986218, BCD-145, ONC-392, CS1002, REGN4659, and ADG116, which are known to those of ordinary skill in the art.

In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibody disclosed in any one of the following patent publications: US 2019/0048096 A1; US 2020/0223907; US 2019/0201334; US 2019/0201334 ; US 2005/0201994; EP 1212422 B1; WO 2018/204760; WO 2018/204760; WO 2001/014424; WO 2004/035607; WO 2006/09649; WO2005092380; WO 2007/123737; WO 2006/029219; WO 2010/0979597; WO 2006/12168; Additional CTLA-4 antibodies are described in: U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; PCT Publication Nos. WO 01/14424 and WO 00/37504; and U.S. Publication No. 2002/0039581 and 2002/086014; and/or US Patent Nos. 5,977,318, 6,682,736, 7,109,003 and 7,132,281, each of which is incorporated herein by reference. In some embodiments, anti-CTLA-4 antibodies such as those disclosed in WO 98/42752; US Pat. Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proceedings of the National Academy of Sciences, 1998 , 95 , 10067-10071 (1998); Camacho et al., Journal of Clinical Oncology 2004 , 22, 145 (Abstract No. 2505 (2004) (antibody CP-675206); or Mokyr et al., Cancer Res. , 1998 , 58, 5301-5304 (1998), each of which is incorporated herein by reference.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand as disclosed in WO 1996/040915 (herein incorporated by reference).

In some embodiments, the CTLA-4 inhibitor is a nucleic acid inhibitor of CTLA-4 expression. For example, anti-CTLA-4 RNAi molecules can be in the form of molecules described in: PCT Publication Nos. WO 1999/032619 and WO 2001/029058; US Publication Nos. 2003/0051263, 2003/0055020 No. 2003/0056235, 2004/265839, 2005/0100913, 2006/0024798, 2008/0050342, 2008/0081373, 2008/0248576 and 2008/055443; And/or US Patent Nos. 6,506,559, 7,282,564, 7,538,095 and 7,560,438 (incorporated herein by reference). In some instances, the anti-CTLA-4 RNAi molecule is in the form of a double-stranded RNAi molecule described in European Patent No. EP 1309726 (herein incorporated by reference). In some instances, the anti-CTLA-4 RNAi molecule is in the form of a double-stranded RNAi molecule described in US Patent Nos. 7,056,704 and 7,078,196 (herein incorporated by reference). In some embodiments, the CTLA-4 inhibitor is an aptamer described in PCT Publication No. WO 2004/081021 (herein incorporated by reference).

In other embodiments, the anti-CTLA-4 RNAi molecules of the present invention are described in U.S. Patent Nos. 5,898,031, 6,107,094, 7,432,249 and 7,432,250 and European Application No. EP 0928290 (incorporated herein by reference) The RNA molecule described in middle). D. Combinations with PD-1, PD-L1 and CTLA-4 inhibitors

In some embodiments, TIL therapy provided to cancer patients may comprise treatment with a therapeutic TIL population alone, or may comprise combination therapy comprising TILs and one or more PD-1 and/or PD-L1 and/or CTLA-4 inhibitors.

In some embodiments, TIL therapy provided to a cancer patient comprises treatment with a combination of a therapeutic TIL population and one or more PD-1 inhibitors. In some embodiments, TIL therapy provided to cancer patients comprises treatment with a combination of a therapeutic TIL population and one or more PD-L1 inhibitors. In some embodiments, TIL therapy provided to a cancer patient comprises treatment with a therapeutic TIL population in combination with one or more CTLA-4 inhibitors. In some embodiments, TIL therapy provided to a cancer patient comprises treatment with a therapeutic TIL population in combination with one or more PD-1 inhibitors and one or more PD-L1 inhibitors. In some embodiments, TIL therapy provided to a cancer patient comprises treatment with a therapeutic TIL population in combination with one or more PD-L1 inhibitors and one or more CTLA-4 inhibitors. In some embodiments, TIL therapy provided to a cancer patient comprises treatment with a therapeutic TIL population in combination with one or more CTLA-4 inhibitors and one or more PD-1 inhibitors. In some embodiments, TIL therapy provided to a cancer patient comprises a combination of a therapeutic TIL population and one or more PD-1 inhibitors, one or more PD-L1 inhibitors, and one or more CTLA-4 inhibitors treat. Any suitable PD-1, PD-L1 or CTLA-4 inhibitor known in the art, such as the inhibitors described herein, can be used. E. Lymphocyte Depleting Preconditioning in Patients

In some embodiments, the invention includes a method of treating cancer with a population of TILs, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the disclosure. In some embodiments, the invention includes TIL populations for use in the treatment of cancer in patients who have been pretreated with nonmyeloablative chemotherapy. In some embodiments, the TIL population is administered by infusion. In some embodiments, the nonmyeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m2/d for 5 days (Days 27 to 23 prior to TIL infusion). In some embodiments, following nonmyeloablative chemotherapy and TIL infusion (Day 0) according to the present disclosure, the patient receives IL-2 (aldesleukin, can be administered intravenously) at 720,000 IU/kg every 8 hours PROLEUKIN commercially available) intravenous infusion to achieve physiological tolerance. In certain embodiments, the population of TILs is used to treat cancer in combination with IL-2, wherein the IL-2 is administered after the population of TILs.

Experimental findings suggest that lymphocyte depletion plays a key role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for elements of the immune system (the 'interleukin pool') prior to adoptive transfer of tumor-specific T lymphocytes. Accordingly, some embodiments of the invention employ a lymphocyte depletion step (sometimes also referred to as "immunosuppressive conditioning") in the patient prior to the introduction of the TILs of the invention.

In general, lymphocyte depletion is achieved using the administration of fludarabine or cyclophosphamide (the active form is called mafosfamide), and combinations thereof. Such methods are described in Gassner et al., Cancer Immunology Immunotherapy 2011 , 60, 75-85, Muranski et al., Nature Clin Practice Oncology , 2006, 3, 668-681, Dudley et al., Journal of Clinical Oncology 2008 , 26, 5233-5239 and Dudley et al., Journal of Clinical Oncology 2005 , 23, 2346-2357, all of which are incorporated herein by reference in their entirety.

In some embodiments, fludarabine is administered at a concentration of 0.5 μg/mL to 10 μg/mL fludarabine. In some embodiments, fludarabine is administered at a concentration of 1 μg/mL fludarabine. In some embodiments, fludarabine is administered for 1, 2, 3, 4, 5, 6, or 7 or more days of treatment. In some embodiments, fludarabine is administered at 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day kg/day, 40 mg/kg/day or 45 mg/kg/day. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 2 to 7 days. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 4 to 5 days. In some embodiments, fludarabine treatment is administered at 25 mg/kg/day for 4 to 5 days.

In some embodiments, the active form of cyclophosphamide, mafosfamide, is obtained at a concentration of 0.5 μg/mL to 10 μg/mL by administering cyclophosphamide. In some embodiments, the active form of cyclophosphamide, mafosfamide, is obtained at a concentration of 1 μg/mL by administering cyclophosphamide. In some embodiments, cyclophosphamide is administered for 1, 2, 3, 4, 5, 6, or 7 or more days of treatment. In some embodiments, cyclophosphamide is formulated at 100 mg/m2/day, 150 mg/m2/day, 175 mg/m2/day, 200 mg/m2/day, 225 mg /m2/day, 250 mg/m2/day, 275 mg/m2/day or 300 mg/m2/day. In some embodiments, cyclophosphamide is administered intravenously (ie, i.v.). In some embodiments, cyclophosphamide treatment is administered at 35 mg/kg/day for 2 to 7 days. In some embodiments, cyclophosphamide treatment is administered intravenously at 250 mg/m2/day for 4 to 5 days. In some embodiments, cyclophosphamide treatment is administered intravenously at 250 mg/m2/day for 4 days.

In some embodiments, lymphocyte depletion is performed by administering fludarabine and cyclophosphamide to the patient together. In some embodiments, fludarabine is administered intravenously at 25 mg/m2/day and cyclophosphamide is administered intravenously at 250 mg/m2/day over 4 days.

In some embodiments, by administering cyclophosphamide at a dose of 60 mg/m/day for two days followed by fludarabine at a dose of 25 mg/m/day for five days Lymphocyte depletion.

In some embodiments, lymphatic monitoring is performed by administering cyclophosphamide at a dose of 60 mg/m2/day for two days and fludarabine at a dose of 25 mg/m2/day for five days. Lymphocyte depletion, where both cyclophosphamide and fludarabine were administered on the first two days, and where lymphocyte depletion was performed for a total of five days.

In some embodiments, cyclophosphamide is administered at a dose of about 50 mg/m2/day for two days and fludarabine at a dose of about 25 mg/m2/day for five days. Lymphocyte depletion was performed in which both cyclophosphamide and fludarabine were administered on the first two days and in which lymphocyte depletion was performed for a total of five days.

In some embodiments, cyclophosphamide is administered at a dose of about 50 mg/m2/day for two days and fludarabine at a dose of about 20 mg/m2/day for five days. Lymphocyte depletion was performed in which both cyclophosphamide and fludarabine were administered on the first two days and in which lymphocyte depletion was performed for a total of five days.

In some embodiments, cyclophosphamide is administered at a dose of about 40 mg/m2/day for two days and fludarabine at a dose of about 20 mg/m2/day for five days. Lymphocyte depletion was performed in which both cyclophosphamide and fludarabine were administered on the first two days and in which lymphocyte depletion was performed for a total of five days.

In some embodiments, cyclophosphamide is administered at a dose of about 40 mg/m2/day for two days and fludarabine at a dose of about 15 mg/m2/day for five days. Lymphocyte depletion was performed in which both cyclophosphamide and fludarabine were administered on the first two days and in which lymphocyte depletion was performed for a total of five days.

In some embodiments, cyclophosphamide is administered at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two consecutive days, followed by 25 Fludarabine was administered at a dose of mg/m2/day for three days for lymphodepletion.

In some embodiments, cyclophosphamide is administered with mesna. In some embodiments, mesna is administered at 15 mg/kg. In some embodiments, mesna is infused, and if infused continuously, over 24 hours, with the start of the respective cyclophosphamide dose, mesna may be infused with cyclophosphamide over approximately 2 hours (Day -5 and/or Day -4), followed by an infusion of 3 mg/kg/hour for the remaining 22 hours.

In some embodiments, the lymphocyte depletion comprises the step of treating the patient with an IL-2 regimen beginning on the day after administration of the third TIL population to the patient.

In some embodiments, the lymphocyte depletion comprises the step of treating the patient with an IL-2 regimen beginning on the day the third TIL population is administered to the patient.

In some embodiments, lymphocyte depletion comprises 5 days of preconditioning therapy. In some embodiments, the number of days is indicated as day -5 to day -1, or day 0 to day 4. In some embodiments, the regimen comprises cyclophosphamide on Day -5 and Day -4 (ie, Day 0 and Day 1). In some embodiments, the regimen comprises intravenous cyclophosphamide on Day -5 and Day -4 (ie, Day 0 and Day 1). In some embodiments, the regimen comprises 60 mg/kg intravenous cyclophosphamide on Day -5 and Day -4 (ie, Day 0 and Day 1). In some embodiments, cyclophosphamide is administered with mesna. In some embodiments, the regimen further comprises fludarabine. In some embodiments, the regimen further comprises intravenous fludarabine. In some embodiments, the regimen further comprises intravenous fludarabine at 25 mg/ ^m2 . In some embodiments, the regimen further comprises intravenous fludarabine at 25 mg/ ^m2 on Day -5 and Day -1 (ie, Day 0 to Day 4). In some embodiments, the regimen further comprises intravenous fludarabine at 25 mg/ ^m2 on Day -5 and Day -1 (ie, Day 0 to Day 4).

In some embodiments, the nonmyeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and cyclophosphamide at 25 mg/m2/day for two consecutive days. Fludarabine was dosed, followed by fludarabine at a dose of 25 mg/m2/day for five days.

In some embodiments, the nonmyeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days, followed by 25 mg/m2/day Fludarabine was dosed for five days.

In some embodiments, the nonmyeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days, followed by 25 mg/m2/day Fludarabine was dosed for three days.

In some embodiments, the nonmyeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and cyclophosphamide at 25 mg/m2/day for two consecutive days. Fludarabine was dosed, followed by fludarabine at a dose of 25 mg/m2/day for three days.

In some embodiments, the nonmyeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and cyclophosphamide at 25 mg/m2/day for two consecutive days. Fludarabine was dosed, followed by fludarabine at a dose of 25 mg/m2/day for one day.

In some embodiments, the nonmyeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days, followed by 25 mg/m2/day Fludarabine was dosed for three days.

In some embodiments, the nonmyeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and cyclophosphamide at 25 mg/m2/day for two consecutive days. Fludarabine was dosed, followed by fludarabine at a dose of 25 mg/m2/day for three days.

In some embodiments, the nonmyeloablative lymphodepleting regimen is administered according to Table 26.

In some embodiments, the nonmyeloablative lymphodepleting regimen is administered according to Table 27.

In some embodiments, the nonmyeloablative lymphodepleting regimen is administered according to Table 28.

In some embodiments, the nonmyeloablative lymphocyte-depleting regimen is administered according to Table 29.

In some embodiments, the nonmyeloablative lymphodepleting regimen is administered according to Table 30.

In some embodiments, the nonmyeloablative lymphocyte-depleting regimen is administered according to Table 31.

In some embodiments, the nonmyeloablative lymphodepleting regimen is administered according to Table 32.

In some embodiments, the nonmyeloablative lymphocyte-depleting regimen is administered according to Table 33.

In some embodiments, the TIL infusions used with the foregoing examples of myeloablative lymphocyte depletion regimens can be any of the TIL compositions described herein, and the addition of IL-2 regimens and administration are as described herein Co-therapy (such as PD-1 and PD-L1 inhibitors). F. IL-2 regimen

In some embodiments, the IL-2 regimen comprises a high-dose IL-2 regimen, wherein the high-dose IL-2 regimen comprises aldesleukin or a biosimilar or variant thereof administered to a therapeutic TIL population for treatment Intravenous administration started on the same day after the effective portion, wherein aldesleukin or its biosimilar or variant system was infused intravenously at 0.037 mg/kg or 0.044 mg/kg IU/kg every eight hours using a 15-minute bolus (patient body weight) until tolerated, up to a maximum of 14 doses. After a 9-day rest, this schedule can be repeated for an additional 14 doses, up to a total of 28 doses. In some embodiments, IL-2 is administered in 1, 2, 3, 4, 5 or 6 doses. In some embodiments, IL-2 is administered in a maximum dose of up to 6 doses.

In some embodiments, the IL-2 regimen comprises a step-down IL-2 regimen. Decreasing IL-2 regimens have been described in O'Day et al., "Journal of Clinical Oncology" 1999 , 17, 2752-61 and Eton et al., "Cancer" 2000, 88, 1703-9, the disclosures of which are in Incorporated herein by reference. In some embodiments, the step-down IL-2 therapy comprises 18×10 ⁶ IU/m ² administered intravenously over 6 hours, followed by 18×10 ⁶ IU/m ² administered intravenously over 12 hours, followed by 24 hours intravenously 18×10 ⁶ IU/m ² was administered internally, followed by 4.5×10 ⁶ IU/m ² aldesleukin or a biosimilar or variant thereof intravenously over 72 hours. This treatment cycle can be repeated every 28 days for a maximum of four cycles. In some embodiments, the step-down IL-2 regimen comprises 18,000,000 IU/m ² on day 1, 9,000,000 IU/m ² on day 2, and 4,500,000 IU/m ² on days 3 and 4.

In some embodiments, the IL-2 regimen comprises a low dose IL-2 regimen. Any low-dose IL-2 regimen known in the art can be used, including Dominguez-Villar and Hafler, Nat. Immunology 2000, 19, 665-673; Hartemann et al., The Lancet Diabetes et al. Low - dose IL- _ _ _ 2, the disclosures of these documents are incorporated herein by reference. In some embodiments, the low-dose IL-2 regimen comprises ^aldesleukin or ^a biosimilar or variant thereof administered as continuous infusion for 5 days every 24 hours; followed by 2- Do not administer IL-2 therapy for 6 days; if necessary, then administer aldesleukin or its biosimilar or variant intravenously for 5 days, with a continuous infusion of 18×10 ⁶ IU/m ² every 24 hours; In the event that IL-2 therapy was not administered for the following 3 weeks, additional cycles could be administered thereafter.

In some embodiments, IL-2 is administered in a maximum dose of up to 6 doses. In some embodiments, high dose IL-2 regimens are suitable for pediatric use. In some embodiments, aldesleukin is used at doses of 600,000 International Units (IU)/kg every 8 to 12 hours for a maximum of 6 doses. In some embodiments, aldesleukin is used at doses of 500,000 international units (IU)/kg every 8 to 12 hours for up to 6 doses. In some embodiments, aldesleukin is used at doses of 400,000 International Units (IU)/kg every 8 to 12 hours for up to 6 doses. In some embodiments, aldesleukin is used at doses of 500,000 international units (IU)/kg every 8 to 12 hours for up to 6 doses. In some embodiments, aldesleukin is used at doses of 300,000 international units (IU)/kg every 8 to 12 hours for up to 6 doses. In some embodiments, aldesleukin is used at doses of 200,000 International Units (IU)/kg every 8 to 12 hours for up to 6 doses. In some embodiments, aldesleukin is used at doses of 100,000 international units (IU)/kg every 8 to 12 hours for up to 6 doses.

In some embodiments, the IL-2 regimen comprises administering pegylated IL-2 at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days. In some embodiments, the IL-2 regimen comprises administering bepideleukin or a fragment, variant thereof, at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days body or biosimilar.

In some embodiments, the IL-2 regimen comprises administering THOR-707, or a fragment, variant, or biosimilars.

In some embodiments, the IL-2 regimen comprises administering Nevanugin alfa or a fragment, variant or biosimilar thereof following administration of TIL. In certain embodiments, naivanugin is administered to the patient at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some embodiments, the IL-2 regimen comprises administering an IL-2 fragment grafted onto the antibody backbone. In some embodiments, the IL-2 regimen comprises administering an antibody cytokine graft protein that binds an IL-2 low affinity receptor. In some embodiments, the antibody cytokine graft protein comprises a heavy chain variable region (V _H ), which comprises complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (V _L ), which comprises LCDR1, LCDR2, LCDR3; and IL-2 molecules or fragments thereof grafted into the CDRs of _VH or _VL , wherein the antibody interleukin graft protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine graft protein comprises a heavy chain variable region (V _H ), which comprises complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (V _L ), which comprises LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into a CDR of a _VH or _VL , wherein the IL-2 molecule is a mutein, and wherein the antibody interleukin graft protein preferentially expands over regulatory T cells T effector cells. In some embodiments, the IL-2 regimen comprises administering the antibody or fragment, variant or biosimilar thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days A substance, the antibody comprising a heavy chain selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:38 and a light chain selected from the group consisting of SEQ ID NO:37 and SEQ ID NO:39.

In some embodiments, the antibody interleukin graft protein described herein has a longer serum half-life than a wild-type IL-2 molecule such as but not limited to aldesleukin (Proleukin®) or a comparable molecule.

In some embodiments, the TIL infusions used with the preceding examples of myeloablative lymphocyte depletion protocols can be any of the TIL compositions described herein and can also include MIL and PBL infusions instead of TIL infusions, and the addition of IL -2 regimen and administration of co-therapy as described herein (such as PD-1 and/or PD-L1 inhibitors and/or CTLA-4 inhibitors). example

The embodiments contemplated herein are now described with reference to the following examples. These examples are provided for purposes of illustration only and the disclosure should in no way be read as limited to these examples, but rather should be understood to cover any and all variations that become apparent as a result of the teachings provided herein. Example 1 : Preparation of medium for PRE-REP and REP processes

This example describes the procedure used to prepare tissue culture media suitable for protocols involving the culture of tumor infiltrating lymphocytes (TILs) derived from various solid tumors. This medium can be used to prepare any of the TILs described in this application and in other examples.

Preparation of CM1 The following reagents were removed from the freezer and allowed to warm in a 37°C water bath: (RPMI1640, human AB serum, 200 mM L-glutamine). Prepare CM1 medium by adding each component on top of a 0.2 µm filter unit appropriate for the volume to be filtered according to Table 34 below. Store at 4 °C.

On the day of use, preheat the required amount of CM1 in a 37°C water bath and add 6000 IU/mL IL-2.

製備 CM2 According to Table 35, additional supplements can be made as needed.

Prepare CM2

Take out the prepared CM1 from the refrigerator or prepare fresh CM1. Remove AIM-V® from the refrigerator, and prepare the required amount of CM2 by mixing the prepared CM1 with an equal volume of AIM-V® in a sterile medium bottle. Add 3000 IU/mL IL-2 to the CM2 medium on the day of use. Make enough CM2 with 3000 IU/mL IL-2 on the day of use. CM2 medium bottles were labeled with their name, initials of the manufacturer, their filtration/preparation date, two week expiration date, and stored at 4°C until needed for tissue culture. Prepare CM3

On the day of use, prepare CM3. CM3 is the same as AIM-V® medium but supplemented with 3000 IU/mL IL-2 on the day of use. Prepare the amount of CM3 required for the experiment by directly adding the IL-2 stock solution to the AIM-V bottle or bag. Mix well by shaking gently. Immediately after adding to AIM-V, label the bottle "3000 IU/mL IL-2". If excess CM3 was present, it was stored in bottles at 4°C, labeled with the name of the medium, the initials of the person who prepared it, the date the medium was prepared and its expiration date (7 days after preparation). After 7 days of storage at 4°C, the medium supplemented with IL-2 was discarded. Prepare CM4

CM4 was the same as CM3 but additionally supplemented with 2 mM GlutaMAX ^™ (final concentration). For every 1 L of CM3, add 10 mL of 200 mM GlutaMAX ^™ . Amounts of CM4 to meet experimental needs were prepared by adding IL-2 stock solution and GlutaMAX ^™ stock solution directly to AIM-V vials or bags. Mix well by shaking gently. Immediately after adding to AIM-V, the bottle was labeled "3000 IL/mL IL-2 with GlutaMAX". If excess CM4 was present, it was stored in bottles at 4°C, labeled with the name of the medium, "GlutaMAX" and its expiration date (7 days after preparation). After storage at 4°C for more than 7 days, the medium supplemented with IL-2 was discarded. Example 2 : Use of IL-2 , IL-15 and IL-21 Cytokines Mixture

This example describes the use of the IL-2, IL-15, and IL-21 cytokines as additional T cell growth factors in combination with the TIL process of any of the examples herein.

Using the procedure described herein, TILs can be grown from tumors in the presence of IL-2 in one group of the experiment, and at the start of the culture, IL-2, IL-15, and IL can be used in another group. -21 combination to replace IL-2. At the completion of pre-REP, cultures were assessed for expansion, phenotype, function (CD107a+ and IFN-γ) and TCR Vβ repertoire. IL-15 and IL-21 are described elsewhere herein and in Santegoets et al., J. Transl. Med. , 2013, 11, 37.

The results can show that enhanced TIL expansion (>20%) in CD4+ and CD8+ cells can be observed under IL-2, IL-15 and IL-21 treatment conditions relative to IL-2 only conditions. In TILs obtained from IL-2, IL-15 and IL-21 treated cultures relative to IL-2 only cultures, there was a skew towards a significant CD8+ population with a skewed TCR Vβ lineage. IFN-γ and CD107a were elevated in TILs treated with IL-2, IL-15 and IL-21 compared to TILs treated with IL-2 alone. Example 3 : Identification of individual batches of gamma- irradiated peripheral monocytes

This example describes a simplified procedure for identifying individual batches of gamma-irradiated peripheral mononuclear cells (PBMCs, also known as monocytes or MNCs) for use as allogeneic feeder cells in the exemplary methods described herein.

Each batch of irradiated MNC feeder cells was prepared from an individual donor. Each lot or donor was individually screened for the ability to expand TILs in REPs in the presence of purified anti-CD3 (cloning OKT3) antibodies and interleukin-2 (IL-2). In addition, each batch of feeder cells was tested without the addition of TIL to verify that the dose of gamma irradiation received was sufficient to render them nonreplicative.

REP of TILs requires gamma-irradiated, growth-arrested MNC feeder cells. Membrane receptors on feeder MNCs bind to anti-CD3 (strain OKT3) antibodies and crosslink to TILs in REP plates, stimulating TIL expansion. Feeder cell batches were prepared from leukapheresis of whole blood obtained from individual donors. Leukapheresis products were centrifuged on Ficoll-Hypaque, washed, irradiated and stored frozen under GMP conditions.

It is important not to infuse live feeder cells into patients receiving TIL therapy, as this may cause graft-versus-host disease (GVHD). Consequently, feeder cells undergo growth arrest upon gamma irradiation of these cells, resulting in double-stranded DNA breaks and loss of cell viability in MNC cells upon re-culture.

Feeder cell batches were evaluated according to two criteria: (1) their ability to expand TILs >100-fold in co-cultures, and (2) their incompetence to replicate.

Feeder cell batches were tested in a mini-REP format using two main pre-REP TIL lines grown in upright T25 tissue culture flasks. The feeder cell batches were tested against two different TIL cell lines, as each TIL line has a unique ability to proliferate in response to activation in REP. As a control, one of the irradiated batches of MNC feeder cells historically shown to meet the above criteria was operated with the test batch.

Sufficient stocks of the same pre-REP TIL line for testing all conditions and all feeder batches are available to ensure that all batches tested in a single experiment are equally tested.

For each batch of feeder cells tested, there were a total of six T25 flasks: Pre-REP TIL Strain #1 (2 flasks); Pre-REP TIL Strain #2 (2 flasks); and Control (2 flasks). Flasks containing TIL lines #1 and #2 were used to assess the ability of feeder cell batches to expand TILs. Feeder control flasks are used to assess the non-replicating capacity of feeder batches. A. Experimental protocol

On day -2/3, thaw the TIL lines. CM2 medium was prepared and CM2 was warmed in a 37°C water bath. Prepare 40 mL of CM2 supplemented with 3000 IU/mL IL-2. Keep warm until ready to serve. 20 mL of pre-warmed CM2 without IL-2 was placed in each of two 50 mL conical tubes and labeled with the name of the TIL strain used. The two indicated pre-REP TIL lines were removed from the LN2 reservoir and vials were transferred to the tissue culture room. Thaw by placing the vial in a 37°C water bath in a zip-sealed storage bag until a small amount of ice remains.

Using a sterile pipette, immediately transfer the contents of each vial to 20 mL of CM2 in the prepared labeled 50 mL conical tube. Cells were washed with IL-2-free CM2 made up to 40 mL and centrifuged at 400×CF for 5 minutes. The supernatant was aspirated and resuspended in 5 mL of warm CM2 supplemented with 3000 IU/mL IL-2.

Take small aliquots (20 µL) in duplicate and perform cell counts using an automated cell counter. record count. While counting, the 50 mL conical tubes with TIL cells were placed in a humidified 37°C, 5% CO2 incubator with the cap loose to allow gas exchange. Cell concentration was determined and TILs were diluted to 1 x ¹⁰⁶ cells/ml in CM2 supplemented with 3000 IU/mL IL-2.

Grow at 2 mL per well in as many wells of 24-well tissue culture plates in a humidified 37°C incubator as needed until day 0 of the mini-REP. Different TIL strains were cultured in separate 24-well tissue culture dishes to avoid confusion and potential cross-contamination.

On day 0, start micro-REP. Prepare enough CM2 medium for the number of feeder cell batches to be tested. (For example, to test 4 feeder cell batches at one time, prepare 800 mL of CM2 medium). A portion of the CM2 prepared above was aliquoted and supplemented with 3000 IU/mL IL-2 for cell culture. (eg, for testing 4 feeder cell batches at once, prepare 500 mL of CM2 medium with 3000 IU/mL IL-2).

Working independently with each TIL strain to prevent cross-contamination, 24-well plates with TIL cultures were removed from the incubator and transferred to BSCs.

Using a sterile pipette or 100-1000 µL pipette and tip, remove approximately 1 mL of medium from each well containing the TIL to be used and place it into an unused well of a 24-well tissue culture dish.

Using a fresh sterile pipette or a 100-1000 µL pipette and tip, resuspend the cells by mixing the remaining medium with the TIL in the well, and then transfer the cell suspension to a 50 oz. mL conical tube and record the volume.

The wells were washed with retained medium and the volume was transferred to the same 50 mL conical tube. Spin the cells at 400×CF to collect the cell pellet. Aspirate the medium supernatant and resuspend the cell pellet in 2-5 mL of CM2 medium containing 3000 IU/mL IL-2, the volume used is based on the number of wells collected and the size of the pellet, i.e. , the volume should be sufficient to ensure a concentration > 1.3 x 10 ⁶ cells/ml.

Using a serological pipette, the cell suspension was mixed well and the volume was recorded. Remove 200 µL and count cells using an automated cell counter. While counting, the 50 mL conical tubes with TIL cells were placed in a humidified 5% CO2, 37°C incubator with the cap loose to allow gas exchange. record count.

The 50 mL conical tube containing TIL cells was removed from the incubator and the cells therein were resuspended at a concentration of 1.3 x ¹⁰⁶ cells/ml in warm CM2 supplemented with 3000 IU/mL IL-2. Place the 50 mL conical tube back into the incubator and loosen the cap.

Repeat the above steps for the second TIL strain.

Just before the TILs were to be spread into the T25 flasks used for the experiment, the TILs were diluted 1:10 as shown below to a final concentration of 1.3 x ¹⁰⁵ cells/ml.

Prepare MACS GMP CD3 pure (OKT3) working solution. OKT3 stock solution (1 mg/mL) was removed from the 4°C freezer and placed in BSC. A final concentration of 30 ng/mL OKT3 was used in medium for mini-REP.

In each T25 flask used for the experiment, 600 ng of OKT3 was required per 20 mL; this corresponds to 60 µL of the 10 µg/mL solution required per 20 mL, or for each feeder cell lot, for all six cultures tested The bottle requires 360 µL.

For each feeder cell batch tested, for a working concentration of 10 µg/mL, prepare 400 µL of a 1:100 dilution of 1 mg/mL OKT3 (e.g., for testing 4 feeder cell batches at once, prepare 1600 µL of 1:100 dilution of 1 mg/mL OKT3: 16 µL of 1 mg/mL OKT3 + 1.584 mL of CM2 medium with 3000 IU/mL IL-2).

Prepare T25 culture flasks. Prior to feeder cell preparation, each flask was labeled and filled with CM2 medium. Place the flask in a humidified 5% _CO2 incubator at 37°C to keep the medium warm while waiting for the remaining components to be added. After the feeder cells were prepared, the components were added to the CM2 in each flask.

Additional information is provided in Table 36.

Prepare feeder cells. For this protocol, at least 78 x ¹⁰⁶ feeder cells were required per batch tested. Each 1 mL vial frozen by SDBB had 100 x ¹⁰⁶ viable cells when frozen. Assuming 50% recovery after thawing from liquid _N2 storage, it is recommended to thaw at least two 1 mL vials of feeder cells per batch, providing an estimated 100 x ¹⁰⁶ viable cells to each REP. Alternatively, if supplied in 1.8 mL vials, only one vial provides sufficient feeder cells.

Prior to thawing the feeder cells, approximately 50 mL of CM2 without IL-2 was pre-warmed for each feeder cell batch to be tested. Remove vials of designated feeder cell batches from LN2 storage, place in zip lock storage bags, and place on ice. Vials were thawed in a closed zip-top storage bag by submersion in a 37°C water bath. Remove the vial from the ziplock bag, spray or wipe with 70% EtOH, and transfer to the BSC.

Using a pipette, immediately transfer the contents of the feeder vial to 30 mL of warmed CM2 in a 50 mL conical tube. The vial was washed with a small volume of CM2 to remove any residual cells in the vial and centrifuged at 400×CF for 5 minutes. The supernatant was aspirated and resuspended in 4 mL of warm CM2 plus 3000 IU/mL IL-2. Remove 200 µL and count cells using an automated cell counter. record count.

Cells were resuspended at 1.3 x ¹⁰⁷ cells/ml in warm CM2 plus 3000 IU/mL IL-2. Dilute TIL cells from 1.3 x ¹⁰⁶ cells/ml to 1.3 x ¹⁰⁵ cells/ml.

Set up co-cultures. Dilute TIL cells from 1.3 x ¹⁰⁶ cells/ml to 1.3 x ¹⁰⁵ cells/ml. Add 4.5 mL of CM2 medium to the 15 mL conical tube. The TIL cells were removed from the incubator and resuspended well using a 10 mL serological pipette. 0.5 mL of cells was removed from the 1.3 x ¹⁰⁶ cells/mL TIL suspension and added to 4.5 mL of medium in a 15 mL conical tube. Return the TIL stock solution vial to the incubator. Mix well. Repeat the above operation for the second TIL strain.

Flasks with pre-warmed media for single feeder cell batches were transferred from the incubator to the BSC. The feeder cells were mixed by pipetting up and down several times with a 1 mL pipette tip, and 1 mL (1.3×10 ⁷ cells) was transferred to each bottle of the feeder cell batch. Add 60 µL of OKT3 working stock solution (10 µg/mL) to each flask. Return the two control flasks to the incubator.

Transfer 1 mL (1.3 x 10 ⁵ ) of each TIL batch to a correspondingly labeled T25 flask. Return the flask to the incubator and incubate upright. Intervention started on day 5. Repeat this procedure for all feeder cell batches tested.

On day 5, the medium was replaced. CM2 was prepared with 3000 IU/mL IL-2. Each flask requires 10 mL. Using a 10 mL pipette, transfer 10 mL of warmed CM2 with 3000 IU/mL IL-2 to each flask. The flasks were returned to the incubator and incubated upright until day 7. Repeat for all feeder cell batches tested.

On day 7, collect. Remove the flask from the incubator and transfer to the BSC, taking care not to disrupt the cell layer on the bottom of the flask. Without disrupting the cells growing on the bottom of the flask, 10 mL of medium was removed from each test flask and 15 mL from each control flask.

Using a 10 mL serological pipette, resuspend the cells in the remaining medium in medium and mix well to break up any cell clumps. After mixing the cell suspension well by pipetting, remove 200 µL for cell counting. TILs were counted using appropriate standard operating procedures in conjunction with automated cell counter equipment. Counts were recorded on Day 7. Repeat this procedure for all feeder cell batches tested.

Feeder control flasks were assessed for non-replicating capacity, and TIL-containing flasks were assessed for fold expansion starting at day 0.

On day 7, continue to operate the feeder cell control bottle until day 14. After the day 7 feeder cell control flasks were counted, 15 mL of fresh CM2 medium containing 3000 IU/mL IL-2 was added to each control flask. The control flasks were returned to the incubator and incubated in an upright position until day 14.

On day 14, the extended non-proliferative phase of the feeder control flask. Remove the flask from the incubator and transfer to the BSC, taking care not to disrupt the cell layer on the bottom of the flask. Approximately 17 mL of medium was removed from each control flask without disrupting the cells growing on the bottom of the flask. Using a 5 mL serological pipette, resuspend the cells in the remaining medium and mix well to break up any cell clumps. Record the volume of each flask.

After mixing the cell suspension well by pipetting, remove 200 µL for cell counting. TILs were counted using appropriate standard operating procedures in conjunction with automated cell counter equipment and the counts were recorded. Repeat this procedure for all feeder cell batches tested. B. Results and Acceptance Criteria Scheme

result. The dose of gamma irradiation is sufficient to render the feeder cells unable to replicate. All batches are expected to meet the evaluation criteria and also demonstrate a reduction in the total number of viable feeder cells remaining on day 7 of REP culture compared to day 0. All feeder cell batches are expected to meet the following evaluation criteria: 100-fold growth expansion of TILs by day 7 of REP culture. Day 14 counts of feeder control flasks are expected to continue the non-proliferative trend seen at day 7.

Acceptance criteria. Each TIL strain replicate for each batch of feeder cells tested met the following acceptance criteria. The acceptance criterion was twofold, as shown in Table 37 below.

To assess whether the irradiation dose was sufficient to render MNC feeder cells replication-incompetent when cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. Anergy was assessed by total viable cell count (TVC) determined by automated cell counter on days 7 and 14 of REP.

The acceptance criterion was "no growth", meaning that at days 7 and 14 there was no increase in the number of total viable cells relative to the initial number of viable cells placed into culture on day 0 of REP.

The ability of feeder cells to support TIL expansion was assessed. TIL growth was measured as the fold expansion of viable cells from the start of culture on REP day 0 to REP day 7. On day 7, TIL cultures achieved a minimal 100-fold expansion (ie, 100-fold greater than the number of total viable TIL cells placed in culture on REP day 0) as assessed by automated cell counting.

Contingency testing of MNC feeder cell batches not meeting acceptance criteria. In the event that an MNC feeder cell lot does not meet any of the acceptance criteria outlined above, the following steps will be taken to retest the lot to rule out simple experimenter error causing this.

If there were two or more satellite test vials remaining from the batch, the batch was retested. If the lot had one or no remaining satellite test vials, the lot was rejected based on the acceptance criteria listed above.

For identification, the batch in question and the control batch must meet the above acceptance criteria. After meeting these criteria, the lot will be released for use. Example 4 : Preparation of IL-2 stock solution

This example describes the process of dissolving purified lyophilized recombinant human interleukin-2 in a stock sample suitable for use in other tissue culture protocols, including all of those described in this application and in the Examples, including those involving These processes used rhIL-2.

program. Prepare a 0.2% acetic acid solution (HAc). Transfer 29 mL of sterile water to a 50 mL conical tube. Add 1 mL of 1 N acetic acid to the 50 mL conical tube. Mix well by inverting the tube 2-3 times. The HAc solution was sterilized by filtration using a Steriflip filter.

Prepare PBS containing 1% HSA. In a 150 mL sterile filter unit, add 4 mL of 25% HSA stock solution to 96 mL of PBS. Filter the solution. Store at 4°C. Complete the form for each vial of rhIL-2 prepared.

A rhIL-2 stock solution ( ^6x106 IU/mL final concentration) was prepared. Each batch of rhIL-2 varies and required information can be found in the manufacturer's Certificate of Analysis (COA), such as: 1) the mass of rhIL-2 per vial (mg), 2) the specific activity of rhIL-2 (IU/ mg) and 3) recommended 0.2% HAc reconstitution volume (mL).

Calculate the volume of 1% HSA required for a rhIL-2 batch using the following formula:

For example, based on the COA of rhIL-2 batch 10200121 (Cellgenix), the specific activity of a 1 mg vial is 25 x ¹⁰⁶ IU/mg. It is recommended to reconstitute rhIL-2 in 2 mL of 0.2% HAc.

Wipe the rubber stopper of the IL-2 vial with an alcohol wipe. Using a 16G needle attached to a 3 mL syringe, inject the recommended volume of 0.2% HAc into the vial. Be careful not to remove the stopper while withdrawing the needle. Invert the vial 3 times and swirl until all powder is dissolved. Carefully remove the stopper and set aside on an alcohol wipe. Add the calculated volume of 1% HSA to the vial.

Store the rhIL-2 solution. For short-term storage (<72 hours), store vials at 4°C. For long-term storage (>72 hours), the vials were aliquoted into smaller volumes and stored in frozen vials at -20°C until ready to use. Avoid freeze/thaw cycles. Expires 6 months after date of manufacture. Rh-IL-2 labels include supplier and catalog number, lot number, expiration date, operator initials, concentration, and aliquot volume. Example 5 : Cryopreservation process

This example describes the cryopreservation process method for TILs prepared according to the procedures described herein using a CryoMed Controlled Rate Freezer Model 7454 (Thermo Scientific).

The following equipment was used: aluminum cassette holder (compatible with CS750 cryo bags), cryo storage box for 750 mL bags, low pressure (22 psi) liquid nitrogen tank, freezer, thermocouple sensor (tape bag), and CryoStore CS750 Freezing Bags (OriGen Scientific).

The freezing process provided self-nucleation at a rate of 0.5°C to -20°C and a cooling rate of 1°C/min to an end point temperature of -80°C. The program parameters are as follows: Step 1—wait at 4°C; Step 2: 1.0°C/min (sample temperature) to -4°C; Step 3: 20.0°C/min (chamber temperature) to -45°C; Step 4 : 10.0°C/min (chamber temperature) to -10.0°C; step 5: 0.5°C/min (chamber temperature) to -20°C; and step 6: 1.0°C/min (sample temperature) to -80 ℃. Example 6 : Tumor Expansion Process Using Defined Media

The processes disclosed above can be performed in place of the CM1 and CM2 media by corresponding defined media (eg, CTS™ OpTmizer™ T Cell Expansion SFM, Thermo Fisher including, for example, DM1 and DM2). Example 7 : Exemplary GEN 2 Preparation of Cryopreserved Cell Therapy

This example describes a cGMP manufacturing of Iovance Biotherapeutics' TIL cell therapy process in G-REX flasks according to current Good Tissue Practices and current Good Manufacturing Practices. This example describes exemplary cGMP manufacturing of TIL cell therapy procedures in G-REX flasks in accordance with current tissue good manufacturing practice and current good manufacturing practice.

Day 0 CM1 medium preparation. In BSC, add reagents to RPMI 1640 media bottles. Add the following reagents to each vial: heat inactivated human AB serum (100.0 mL); GlutaMax™ (10.0 mL); gentamycin sulfate, 50 mg/mL (1.0 mL); 2-mercaptoethanol (1.0 mL)

Remove unnecessary material from BSC. Media reagents were distributed from the BSC, and Gentamycin Sulfate and HBSS were kept in the BSC for preparation of wash media.

Thaw IL-2 aliquots. Thaw a 1.1 mL aliquot of IL-2 (6×10 ⁶ IU/mL) (BR71424) until all ice has melted. Record IL-2: Batch number and expiry date

Transfer IL-2 stock solution to culture medium. In BSCs, transfer 1.0 mL of IL-2 stock solution to the prepared CM1 day 0 medium bottle. Add 1 bottle of CM1 day 0 medium and 1.0 mL of IL-2 (6×10 ⁶ IU/mL).

Pass the G-REX100MCS into the BSC. G-REX100MCS (W3013130) was aseptically delivered into BSCs.

Pump all complete CM1 Day 0 media into the G-REX100MCS flask. Tissue Fragment Conical Tube or GRex100MCS.

Day 0 tumor wash medium preparation. In BSC, add 5.0 mL of gentamycin (W3009832 or W3012735) to a 1×500 mL bottle of HBSS medium (W3013128). Add to each bottle: HBSS (500.0 mL); Gentamycin Sulfate, 50 mg/ml (5.0 mL). Filtered HBSS containing gentamycin was prepared through a 1 L 0.22 micron filter unit (W1218810).

Day 0 tumor treatment. Tumor samples were obtained and immediately transferred to kits at 2-8°C for processing. Aliquot tumor wash medium. Tumor wash 1 was performed using 8'' forceps (W3009771). Tumors were removed from the vials and transferred to prepared "Wash 1" dishes. This was followed by tumor wash 2 and tumor wash 3. Tumors are measured and assessed. Assess whether >30% necrosis of the total tumor area and/or adipose tissue is observed. When applicable, perform a cleansing split. If the tumor is large and >30% of the tissue surface is observed to be necrotic/fatty, "clear segmentation" is performed by using a combination of scalpel and/or forceps to remove the necrotic/fatty tissue while preserving the tumor internal structure. Segment the tumor. Using a combination of scalpel and/or forceps, the tumor sample is cut into uniform, appropriately sized fragments (up to 6 intermediate fragments). Metastasis of intermediate tumor fragments. Tumor fragments were divided into small pieces approximately 3 x 3 x 3 mm in size. Store intermediate pieces to prevent dehydration. Repeat for middle segment segmentation. The number of small pieces collected was determined. If only desired tissue is retained, additional favorable tumor fragments are selected from the "favorable intermediate fragments" 6-well plate to fill the discard fragments, up to a maximum of 50 fragments.

Prepare conical tubes. Transfer the tumor pellet to a 50 mL conical tube. Prepare BSC for G-REX100MCS. Take out the G-REX100MCS from the incubator. Aseptically transfer the G-REX100MCS flask into the BSC. Tumor fragments were added to G-REX100MCS flasks. Distribute small pieces evenly.

Cultivate G-REX100MCS according to the following parameters: Cultivate G-REX culture bottle: temperature LED display: 37.0±2.0°C; CO ₂ percentage: 5.0±1.5% CO ₂ . Calculation: cultivation time; lower limit = cultivation time + 252 hours; upper limit = cultivation time + 276 hours.

Upon completion of the process, any remaining warmed medium was discarded and an aliquot of IL-2 was thawed.

Day 11 - Media preparation. Monitor the incubator. Incubator parameters: Temperature LED display: 37.0±2.0°C; CO2 percentage: 5.0±1.5% CO2.

Heat 3×1000 mL RPMI 1640 medium (W3013112) bottles and 3×1000 mL AIM-V (W3009501) bottles in the incubator for ≥30 minutes. Remove the RPMI 1640 medium bottle from the incubator. Prepare RPMI 1640 medium bottles. Filter medium. A 3 x 1.1 mL aliquot of IL-2 (6 x ¹⁰⁶ IU/mL) (BR71424) was thawed. Remove the AIM-V medium from the incubator. Add IL-2 to AIM-V. Aseptically transfer the 10 L Labtainer bag and relay pump transfer device into the BSC.

Prepare a 10 L Labtainer medium bag. Prepare the Baxa pump. Prepare a 10L Labtainer medium bag. Pump the medium into a 10 L Labtainer. Remove the autopump straw from the Labtainer bag.

mixed media. Gently rub bag to mix. Sampling of medium according to the sampling plan. Remove 20.0 mL of medium and place in a 50 mL conical tube. Prepare cell counting dilution tubes. In BSC, add 4.5 mL of AIM-V medium labeled "For Cell Count Dilution" and lot number to four 15 mL conical tubes. Reagents were transferred from the BSC to 2 to 8°C. Prepare a 1 L transfer pack. Outside the BSC, weld the 1 L transfer pack (per procedure note 5.11) to the transfer device attached to the prepared "Complete CM2 Day 11 Medium" bag. Prepare feeder cell transfer packs. Incubate complete CM2 day 11 medium.

Day 11 - TIL collection. Preprocess the form. Incubator parameters: Temperature LED display: 37.0±2.0°C; CO ₂ percentage: 5.0±1.5% CO ₂ . Take out the G-REX100MCS from the incubator. Prepare 300 mL transfer packs. Weld the transfer bag to the G-REX100MCS.

Flasks were prepared for TIL collection and TIL collection was initiated. Collect TILs. Using the GatheRex, transfer the cell suspension through a hemofilter into a 300 mL transfer bag. Check for adherent cells on the membrane.

Rinse the flask membrane. Close the clip on the G-REX100MCS. Make sure all clips are closed. Heat seal the TIL and "supernatant" transfer pack. Calculate the volume of the TIL suspension. Prepare supernatant transfer packs for sampling.

A Bac-T sample was drawn. In the BSC, aspirate approximately 20.0 mL of supernatant from the 1 L "supernatant" transfer pack and dispense into sterile 50 mL conical tubes.

BacT was inoculated according to the sampling plan. A 1.0 mL sample was withdrawn from the prepared 50 mL conical tube labeled BacT using an appropriately sized syringe and inoculated into an anaerobic bottle.

Cultivate TILs. Place the TIL transfer pack in the incubator for use. Perform cell counts and calculations. Determine the mean viable cell concentration and mean viability of cells for which cell counts are performed. Survival rate ÷ 2. Viable cell concentration ÷2. Determine the upper and lower limits of the count. Lower limit: mean value of viable cell concentration × 0.9. Upper limit: the average concentration of viable cells × 1.1. Confirm that both counts are within acceptable limits. The average viable cell concentration was determined from all four counts performed.

Adjusted volume of TIL suspension: Calculate the adjusted volume of TIL suspension after removal of the cytometry sample. Total TIL cell volume (A). Adjust the total volume of TIL cells C = A - B with the volume of the cell count sample taken (4.0 mL) (B).

Count the total number of viable TIL cells. Mean viable cell concentration*: total volume; total viable cell number: C=A×B.

Calculations for Flow Cytometry: If the total viable TIL cell count is > 4.0 x 10 ⁷ , calculate the volume of 1.0 x 10 ⁷ cells for flow cytometry samples.

The total number of viable cells required for flow cytometry: 1.0×10 ⁷ cells. Cell volume required for flow cytometry: Divide the viable cell concentration by 1.0×10 ⁷ cells A.

Calculate the volume of TIL suspension equal to 2.0 x ¹⁰⁸ viable cells. As needed, calculate the excess TIL cell volume to be removed, and remove excess TIL and place TIL in an incubator as needed. The total amount of excess TIL removed as needed was calculated.

The calculated amount of CS-10 medium added to excess TIL cells at the target cell concentration for freezing was 1.0 x ¹⁰⁸ cells/ml. Excess TIL was centrifuged as needed. Observe the conical tube and add CS-10.

Fill the vial. Aliquot 1.0 mL of the cell suspension into appropriately sized cryovials. Aliquot remaining volume into appropriately sized cryovials. If the volume is ≤0.5 mL, add CS10 to the vial until the volume is 0.5 mL.

Calculate the cell volume required to obtain 1 x ¹⁰⁷ cells for cryopreservation. Remove samples for cryopreservation. Place TILs in an incubator.

Freezing of samples. Observe the conical tube and slowly add CS-10 and record the volume at which 0.5 mL of CS10 was added.

Day 11 - Feeder cells. Obtain feeder cells. Obtain at least 3 bags of feeder cells from two different batches from the LN2 freezer. Store cells on dry ice until ready to thaw. Prepare a water bath or cryotherm cryogenic storage system. Thaw the feeder cells in a 37.0±2.0°C water bath or cytotherm cryogenic storage system for about 3 to 5 minutes or until the ice just disappears. Remove the medium from the incubator. Pool thawed feeder cells. Add feeder cells to transfer pack. The feeder cells are dispensed from the syringe into the transfer pack. Pooled feeder cells were mixed and transfer packets were labeled.

Calculate the total volume of feeder cell suspension in the transfer pack. Remove the sample for cell counting. Using a separate 3 mL syringe for each sample, aspirate 4 x 1.0 mL cytometry samples from the feeder cell suspension transfer pack using the needle-free injection port. Aliquot each sample into labeled cryovials. A cell count is performed and the decision multiplier is selected and the multiplier is entered. Determine the mean viable cell concentration and mean viability of the performed cell counts. Determine the upper and lower limits of the count and confirm that they are within the limits.

Adjust the volume of the feeder cell suspension. Calculate the adjusted volume of the feeder cell suspension after removal of the cytometry sample. Count the total number of live feeder cells. Obtain additional feeder cells as needed. Thaw additional feeder cells as needed. Place the 4th feeder cell bag in a zipper bag and thaw in a 37.0±2.0°C water bath or cytotherm cryogenic storage system for about 3 to 5 minutes and pool additional feeder cells. Measure the volume. Measure the volume of feeder cells in the syringe and record below (B). Calculate the new total volume of feeder cells. Add feeder cells to transfer pack.

As needed, prepare dilutions by adding 4.5 mL of AIM-V medium to four 15 mL conical tubes. Prepare to count the number of cells. Using a separate 3 mL syringe for each sample, withdraw 4 x 1.0 mL cell count samples from the feeder cell suspension transfer pack using the optional injection port. Perform cell counts and calculations. Determine the average viable cell concentration from all four counts performed. The volume of the feeder cell suspension was adjusted, and the adjusted volume of the feeder cell suspension after removal of the cell count sample was calculated. The total volume of feeder cells minus 4.0 mL removed. Calculate the volume of feeder cell suspension required to obtain 5×10 ⁹ viable feeder cells. Calculate the excess feeder cell volume. Calculate the volume of excess feeder cells to be removed. Remove excess feeder cells.

Using a 1.0 mL syringe with a 16G needle, draw up 0.15 mL of OKT3 and add OKT3. Heat seal the feeder cell suspension transfer pack.

Day 11 G-REX filling and inoculation set up G-REX500MCS. Remove the "Complete CM2 Day 11 Medium" from the incubator and pump the medium into the G-REX500MCS. Pump 4.5 L of culture medium into G-REX500MCS and fill to the line marked on the culture bottle. Heat seal and keep flasks warm as needed. The feeder cell suspension transfer package was welded to the G-REX500MCS. Add feeder cells to G-REX500MCS. heat seal. The TIL suspension transfer pack was welded to the culture flask. Add TIL to G-REX500MCS. heat seal. Incubate the G-REX500MCS at 37.0±2.0°C with the following CO2 percentage: 5.0±1.5% CO2.

Calculate insulation range. Calculations were performed to determine the appropriate time to remove the G-REX500MCS from the incubator on day 16. Lower limit: holding time + 108 hours. Upper limit: holding time + 132 hours.

On day 11 excess TIL was cryopreserved. Intended use: Freeze excess vials of TIL. Verify that CRF is set prior to freezing. Keep frozen. Transfer the vials from the rate controlled freezer to appropriate storage. After freezing is complete, transfer the vials from the CRF to an appropriate storage container. Transfer vial to appropriate storage. The storage location of the record in LN2.

Day 16 Media preparation. Prewarm AIM-V medium. The time to warm the medium was calculated for media bags 1, 2 and 3. Make sure all bags have been warmed up for a duration of 12 to 24 hours. Set up a 10L Labtainer for the supernatant. Attach the larger diameter end of the fluid pump transfer device to one of the female ports of the 10L Labtainer bag using a Luer connector. Set up 10L Labtainer for supernatant and label. Set up a 10L Labtainer for the supernatant. Make sure to close all clips before removing from the BSC. Note: Use the supernatant bag during TIL collection, which can be done in parallel with media preparation.

Thaw IL-2. Thaw 5 x 1.1 mL IL-2 aliquots (6 x ¹⁰⁶ IU/mL) per bag of CTS AIM V medium (BR71424) until all ice has melted. Aliquot 100.0 mL of GlutaMax™. Add IL-2 to GlutaMax™. Prepare the CTS AIM V medium bag for preparation. Prepare the CTS AIM V medium bag for preparation. Multi-stage Baxa pumps. Prepare the culture medium. Pump GlutaMax™ + IL-2 into the bag. Monitoring parameters: Temperature LED display: 37.0±2.0°C, CO ₂ percentage: 5.0±1.5% CO ₂ . Allow complete CM4 day 16 medium to warm. Prepare dilutions.

On the 16th day, REP was divided into bottles. Monitoring incubator parameters: Temperature LED display: 37.0±2.0°C; CO ₂ percentage: 5.0±1.5% CO ₂ . Take out the G-REX500MCS from the incubator. A 1 L transfer pack was prepared and labeled TIL Suspension and weighed to 1 L.

The volume of G-REX500MCS is reduced. Approximately 4.5 L of culture supernatant was transferred from the G-REX500MCS to the 10L Labtainer.

Prepare culture flasks for TIL collection. After removing the supernatant, close all clamps leading to the red line.

Initiate TIL collection. Tap the flask vigorously and swirl the medium to detach the cells to ensure that all cells are detached.

TIL collection. Loosen all clips leading to the TIL suspension transfer bag. Using GatheRex, transfer the cell suspension to the TIL suspension transfer kit. Note: Make sure to maintain the edge slope until all cells and media are collected. Check for adherent cells on the membrane. Rinse the flask membrane. Close the clip on the G-REX500MCS. Heat seal the transfer package containing the TIL. Heat seal the 10L Labtainer containing the supernatant. Record the weight of the transfer bag containing the cell suspension and calculate the suspension volume. Prepare a transfer bag for sample retrieval. Test samples were removed from the cell supernatant.

Sterility and BacT test sampling. Take 1.0 mL sample from the prepared 15 mL labeled BacT conical tube. Remove the sample for cell counting. In the BSC, 4 x 1.0 mL cytometry samples were removed from the "TIL Suspension" transfer pack using a separate 3 mL syringe for each sample.

Remove the mycoplasma sample. Using a 3 mL syringe, remove 1.0 mL from the TIL Suspension Transfer Pack and place in the prepared 15 mL conical tube labeled "Mycoplasma Diluent".

Prepare the transfer pack for vaccination. Place TILs in an incubator. Cell suspensions were removed from the BSCs and placed in an incubator until needed. Perform cell counts and calculations. Cell count samples were first diluted by adding 0.5 mL of the cell suspension to 4.5 mL of the prepared AIM-V medium, resulting in a 1:10 dilution. Determine the mean viable cell concentration and mean viability of the performed cell counts. Determine the upper and lower limits of the count. NOTE: Dilution can be adjusted based on expected cell concentration. The average viable cell concentration was determined from all four counts performed. Adjust the volume of the TIL suspension. Calculate the adjusted volume of the TIL suspension after removal of the cytometry sample. Total TIL cell volume minus 5.0 mL removed for testing.

Count the total number of viable TIL cells. Calculate the total number of flasks to be inoculated. Note: The maximum number of G-REX500MCS flasks to be inoculated is five. If the calculated number of flasks to be inoculated exceeds five, use all available volumes of cell suspension to inoculate only five flasks.

Calculate the number of flasks used for subculture. Calculate the number of media bags needed in addition to the bags prepared. It is calculated that one 10 L bag of "CM4 day 16 medium" should be prepared for every two G-REX-500M culture bottles. Continue to inoculate the first GREX-500M flask while additional medium is prepared and allowed to warm. The calculated number of additional medium bags identified were prepared and allowed to warm up. Fill the G-REX500MCS. Prepare to pump medium and pump 4.5 L of medium into the G-REX500MCS. heat seal. Repeat filling. Keep the flask warm. Calculate the target volume of TIL suspension to add to new G-REX500MCS flasks. If the calculated number of flasks exceeds five, all volumes of cell suspension are used to inoculate only five flasks. Prepare culture flasks for inoculation. Take out the G-REX500MCS from the incubator. Prepare G-REX500MCS for pumping. Close all clamps except the larger filter line. Remove the TIL from the incubator. Prepare cell suspension for inoculation. Aseptically weld the "TIL Suspension" transfer pack (per procedure note 5.11) to the pump inlet line. Place the bag of TIL suspension on the scale.

Inoculate the flask with the TIL suspension. Pump the calculated volume of TIL suspension into the culture flask. heat seal. Fill remaining flask.

Monitor the incubator. Incubator parameters: Temperature LED display: 37.0±2.0°C; CO ₂ percentage: 5.0±1.5% CO ₂ . Keep the flask warm.

Determine the time frame for removing the G-REX500MCS from the incubator on day 22.

Day 22 Wash buffer preparation. Prepare 10L Labtainer bags. In the BSC, the 4'' plasma transfer device was attached to the 10L Labtainer bag via a Luer connector. Prepare 10L Labtainer bags. Close all clamps before transferring out of the BSC. Note: Prepare a 10L Labtainer bag for every two G-REX500MCS culture bottles to be collected. The Plasmalyte was pumped into the 3000 mL bag and the air was removed from the 3000 mL Origen bag by inverting the pump and manipulating the position of the bag. Add 25% human albumin to the 3000 mL bag. Obtain a final volume of 120.0 mL of 25% human albumin.

Prepare IL-2 diluent. Using a 10 mL syringe, withdraw 5.0 mL of LOVO Wash Buffer using the needle-free injection port on the LOVO Wash Buffer bag. Dispense LOVO Wash Buffer into 50 mL conical tubes.

Aliquot the CRF blank bag with LOVO wash buffer. Using a 100 mL syringe, draw 70.0 mL of LOVO Wash Buffer from the needle-free injection port.

Thaw a 1.1 mL aliquot of IL-2 (6 x 106 IU/mL) until all ice has melted. Add 50 µL of IL-2 stock solution (6×10 ⁶ IU/mL) to the 50 mL conical tube labeled "IL-2 Diluent".

Prepare for cryopreservation. Five freezer boxes were placed at 2 to 8°C to precondition them for final product cryopreservation.

Prepare cell counting dilutions. In BSC, add 4.5 mL of AIM-V medium labeled "for cell count dilution" and the lot number to four 15 mL conical tubes. Prepare to count the number of cells. Label the 4 frozen vials with the vial number (1 to 4). Save the vial at the BSC for use.

Day 22 TIL collection. Monitor the incubator. Incubator parameters: Temperature LED display: 37±2.0°C, CO2 percentage: 5%±1.5%. Take out the G-REX500MCS culture bottle from the incubator. Prepare and label TIL collection bags. Close additional connectors. Volume reduction: Transfer approximately 4.5 L of supernatant from the G-REX500MCS to the supernatant bag.

Prepare culture flasks for TIL collection. Initiate TIL collection. Tap the flask vigorously and swirl the medium to dislodge the cells. Make sure all cells are exfoliated. Initiate TIL collection. Release all clips leading to TIL suspension collection bag. TIL collection. Using GatheRex, transfer the TIL suspension to a 3000 mL collection bag. Check for adherent cells on the membrane. Rinse the flask membrane. Close the clips on the G-Rex500MCS, making sure all clips are closed. Transfer the cell suspension to the LOVO source bag. Close all clips. heat seal. Take 4 x 1.0 mL cell count samples

Perform a cell count. Cell counts and calculations were performed using NC-200 and procedure note 5.14. Cell count samples were first diluted by adding 0.5 mL of cell suspension to the prepared 4.5 mL of AIM-V medium. A 1:10 dilution is obtained. The mean viability, viable cell concentration, and total nucleated cell concentration were determined resulting in qualitative cell counts. Determine the upper and lower limits of the count. The mean viability, viable cell concentration, and total nucleated cell concentration were determined resulting in qualitative cell counts. Weigh the LOVO source bag. Count the total number of viable TIL cells. Count the total number of nucleated cells.

Prepare mycoplasma diluent. 10.0 mL was removed from one supernatant bag via the luer sample port and placed in a 15 mL conical tube.

Perform the "TIL G-REX collection" protocol and determine the final product target volume. Load the disposable set. Remove the filtrate bag. Enter the filtrate volume. Place the filtrate container on the bench. Attach PlasmaLyte. Confirmed that PlasmaLyte is attached and observed that PlasmaLyte is moving. Attach the source container to the conduit and verify that the source container is attached. Confirm that PlasmaLyte is moving.

Final dispensing and filling. Target volume/bag calculation. Calculate the volume of CS-10 and LOVO wash buffer to be prepared in the blank bag. Prepare CRF blank bags.

Calculate the volume of IL-2 to be added to the final product. Desired final IL-2 concentration (IU/mL) - 300 IU/mL. IL-2 working stock solution: 6×10 ⁴ IU/mL. Assemble the connected device. Aseptically weld 4S-4M60 to CC2 unit connector. Aseptically weld CS750 freezer bags (according to Procedure Note 5.11) to the prepared bundle. The CS-10 bag was welded onto the tip of the 4S-4M60. TILs were prepared in the presence of IL-2. Using an appropriately sized syringe, the measured amount of IL-2 was withdrawn from the "IL-2 6 x ¹⁰⁴ " aliquot. Label the reconstituted TIL bag. Add the formulated TIL bag to the device. Add CS10. Switch syringes. Aspirate approximately 10 mL of air into the 100 mL syringe and replace the 60 mL syringe on the device. Add CS10. Prepare CS-750 bags. Allocate cells.

Air was removed from the final product bag and a retentate was obtained. Once the last end product bag has been filled, all clips are closed. Aspirate 10 mL of air into a new 100 mL syringe and replace the syringe on the device. Dispense the retentate into 50 mL conical tubes, and label the tube "Retentate" and the lot number. Repeat the air removal step for each bag.

Prepare the final product for cryopreservation, including visual inspection. Store freezer bags on cooling packs or at 2 to 8°C prior to cryopreservation.

Remove the sample for cell counting. Using an appropriately sized pipette, remove 2.0 mL of the retentate and place in a 15 mL conical tube for cell counting. Perform cell counts and calculations. Note: Only dilute one sample to the appropriate dilution to verify that the dilution is sufficient. Additional samples were diluted to the appropriate dilution factor and counts continued. Determine the mean viable cell concentration and mean viability of cells for which cell counts are performed. Determine the upper and lower limits of the count. Note: The dilution can be adjusted based on the expected cell concentration. The average concentration of living cells and the average survival rate were determined. Determine the upper and lower limits of the count. Calculate IFN-γ. Heat seal the final product bag.

Label and collect samples according to the following exemplary sampling plan.

Sterility and BacT tests. Test sampling. In BSC, a 1.0 mL sample was taken from the retained cell suspension collected using an appropriately sized syringe and inoculated anaerobic vials. Repeat the above operation for the aerobic bottle.

Final product cryopreservation prepares rate-controlled freezers (CRF). Verify that CRF is set. Set up the CRF probe. Place the final product and samples in the CRF. Measure the time required to reach 4°C ± 1.5°C and proceed with the CRF run. Complete the CRF and save. Stop CRF when finished running. Remove the box and vial from the CRF. Transfer boxes and vials to vapor phase LN2 for storage. Record storage location.

Post-processing and analysis of the final drug product includes the following tests: (Day 22) CD3+ cells of Day 22 REP by flow cytometry; (Day 22) Gram stain (GMP); (Day 22) Bacterial endotoxin testing by gel clot LAL assay (GMP); (Day 16) BacT sterility assay (GMP); (Day 16) Mycoplasma DNA detection by TD-PCR (GMP); Acceptable appearance attributes; (Day 22) BacT sterility assay (GMP) (Day 22); (Day 22) IFN-γ analysis. TIL production was also analyzed using other potency assays as described herein. Example 8: Exemplary Embodiment of the GEN 3 Amplification Platform Day 0

Prepare tumor wash medium. Warm the medium before starting. Add 5 mL of Gentamycin (50 mg/mL) to a 500 mL HBSS bottle. Add 5 mL of Tumor Wash Medium to a 15 mL Erlenmeyer flask for OKT3 dilution. Prepare feeder cell bags. The feeder cells were aseptically transferred to feeder cell bags and stored at 37°C until use or frozen. If at 37°C, count the feeder cells. If frozen, thaw and then count feeder cells.

The optimal range of feeder cell concentration is between 5×10 ⁴ and 5×10 ⁶ cells/ml. Prepare four conical tubes with 4.5 mL of AIM-V. For each cell count, add 0.5 mL of the cell fraction. If the total number of live feeder cells is ≥1×10 ⁹ cells, continue to adjust the feeder cell concentration. The volume of feeder cells removed from the first feeder cell bag was calculated so that 1 x ¹⁰⁹ cells were added to the second feeder cell bag.

Using a p1000 micropipette, transfer 900 µL of tumor wash medium into OKT3 aliquots (100 µL). Using a syringe and aseptic technique, draw up 0.6 mL of OKT3 and add to the second feeder bag. Adjust medium volume to a total volume of 2 L. Transfer the second bag of feeder cells to the incubator.

OKT3 formulation details: OKT3 can be aliquoted in vials at the original stock concentration (1 mg/mL) in 100 µL aliquots and frozen. Aliquot approximately 10X per 1 mL vial. Store at -80°C. Day 0: 15 micrograms/vial, ie, 30 ng/mL in 500 mL, 1 aliquot up to approximately 60 µL.

Add 5 mL of tumor wash medium to all wells of the 6-well plate marked as excess tumor pellet. Save the tumor wash medium for further use to keep the tumor hydrated during dissection. 50 mL of tumor wash medium was added to each 100 mm petri dish.

Using a ruler as a reference under the dissecting Petri dish lid, segment the tumor into 27 mm ³ -segments (3 x 3 x 3 mm). Divide the middle segment until you reach 60 segments. The total number of final fragments was counted according to the number of final fragments produced (typically 60 fragments per flask) and G-REX-100MCS flasks were prepared.

Favored tissue segments are retained in conical tubes labeled Segment Tube 1 to Segment Tube 4 . The number of G-REX-100MCS flasks to be inoculated with the feeder cell suspension was counted according to the originating fragment tube.

The feeder cell bag was removed from the incubator and inoculated with G-REX-100MCS. Labeled D0 (Day 0).

Tumor fragments were added to cultures in G-REX-100 MCS. Under sterile conditions, unscrew the cap of the G-REX-100MCS marked with tumor fragment culture (D0) 1 and the 50 mL conical tube marked with the fragment tube. Unscrew the open Fragment Tube 1 while gently lifting the cap of the G-REX100MCS. While swirling, medium with fragments was added to the G-REX100MCS. Record the number of segments transferred to G-REX100MCS.

Once the fragments were at the bottom of the GREX flask, 7 mL of medium was aspirated and seven 1 mL aliquots were generated, 5 mL for extended characterization and 2 mL for sterile samples. Five aliquots (final fragment culture supernatant) were stored at -20°C for extended characterization until required.

Inoculate one anaerobic BacT/Alert bottle and one aerobic BacT/Alert bottle with 1 mL of the final fragment culture supernatant. Repeat the above operation for each culture flask to be sampled.

Day 7-8

Prepare feeder cell bags. When frozen, thaw feeder cell bags in a 37°C water bath for 3-5 minutes. If frozen, count feeder cells. The optimal range of feeder cell concentration is between 5×10 ⁴ and 5×10 ⁶ cells/ml. Prepare four conical tubes with 4.5 mL of AIM-V. For each cell count, add 0.5 mL of the cell fraction to a new cryovial tube. Samples were mixed well and cell counts were performed.

If the total number of live feeder cells is ≥2×10 ⁹ cells, proceed to the next step to adjust the feeder cell concentration. The volume of feeder cells removed from the first feeder cell bag was calculated so that 2 x ¹⁰⁹ cells were added to the second feeder cell bag.

Using a p1000 micropipette, transfer 900 µL HBSS into 100 µL OKT3 aliquots. Mix by pipetting up and down 3 times. Prepare two vials.

OKT3 formulation details: OKT3 can be aliquoted in vials at the original stock concentration (1 mg/mL) in 100 µL aliquots and frozen. Approximately 10X aliquots per 1 mL vial. Store at -80°C. Day 7/8: 30 µg/vial, ie, 60 ng/mL in 500 mL, up to 120 µl, in 2 aliquots.

Using a syringe and aseptic technique, draw up 0.6 mL of OKT3 and add to the feeder bag, making sure to add all. Adjust medium volume to a total volume of 2 L. Repeat with the second OKT3 aliquot and add to the feeder bag. Transfer the second bag of feeder cells to the incubator.

Prepare G-REX100MCS flasks with feeder cell suspension. The number of G-REX-100MCS bottles to be processed was recorded against the number of G-REX bottles produced on Day 0. Remove the G-REX flask from the incubator and remove the second feeder cell bag from the incubator.

Remove the supernatant before adding the feeder cell suspension. Attach a 10 mL syringe to the G-REX100 flask and draw 5 mL of medium. Generate five 1 mL aliquots: 5 mL for extended characterization and store 5 aliquots for extended characterization (final fragment culture supernatant) at -20°C until trial commissioner Request. Label each G-REX100 flask and repeat the above operation.

Depending on the number of flasks, take 5-20 x 1 mL samples for characterization : 5 mL = 1 flask 10 mL = 2 flasks 15 mL = 3 flasks 20 mL = 4 cultures bottle

Continue to inoculate feeder cells into G-REX100 MCS, and repeat the above operation for each G-REX100 MCS culture flask. Using the aseptic transfer method, transfer 500 mL of the second feeder cell bag by weight (assuming 1 g = 1 mL) to each G-REX-100MCS culture flask by gravity and record the amount. Label as day 7 culture and repeat for each G-REX100 flask. Transfer the G-REX-100MCS flask to the incubator. Day 10-11 _

Remove the first G-REX-100MCS flask and using aseptic conditions, use a 10 mL syringe to remove 7 mL of the pretreated culture supernatant. Seven 1 mL aliquots were created, 5 mL for extended characterization and 2 mL for sterile samples.

Carefully mix the flask and withdraw 10 mL of the supernatant using a new 10 mL syringe and transfer to a 15 mL tube labeled D10/11 Mycoplasma Supernatant.

Mix the bottles carefully and use a new syringe to withdraw the following volumes according to the number of bottles to be processed : 1 bottle = 40 mL 2 bottles = 20 mL/vial 3 bottles = 13.3 mL/ vial 4 culture flasks = 10ml/bottle

A total of 40 mL should be aspirated from all flasks and pooled in a 50 mL conical tube labeled "Day 10/11 QC Sample" and stored in an incubator until needed. Perform a cell count and dispense cells.

Five aliquots (final fragment culture supernatant) were stored at <-20°C for extended characterization until required. One anaerobic BacT/Alert bottle and one aerobic BacT/Alert bottle were each inoculated with 1 mL of the pretreated culture supernatant.

Continue transferring the cell suspension to the G-REX-500MCS and repeat for each G-REX-100MCS. Using aseptic conditions, transfer the contents of each G-REX-100MCS to the G-REX-500MCS, monitoring the fluid transfer volume of approximately 100 mL at a time. The transfer was stopped when the volume of G-REX-100MCS was reduced to 500 mL.

During the transfer step, use a 10 mL syringe and draw 10 mL of the cell suspension from the G-REX-100MCS into the syringe. According to the number of culture flasks in the culture, follow the instructions to operate. If only 1 flask: Use two syringes to withdraw a total of 20 mL. If there are 2 culture flasks: remove 10 mL from each culture flask. If there are 3 flasks: remove 7 mL from each flask. If there are 4 flasks: remove 5 mL from each flask. Transfer the cell suspension to a common 50 mL conical tube. Keep in the incubator until the cell counting step and QC samples. The total number of cells required for QC is approximately 20e6 cells: 4 x 0.5 mL cell count (cell count initially undiluted).

The number of cells required for the assay is as follows: 1. A minimum of 10 x ¹⁰⁶ cells for potency assays, such as those described herein, or for IFN-γ or granzyme B assays 2. 1 x ¹⁰⁶ cells for In Mycoplasma 3.5×10 ⁶ cells for flow cytometry against CD3+/CD45+

Transfer the G-REX-500MCS culture flask to the incubator.

Prepare QC samples. In this example, the assay requires at least 15 x 10 ⁸ cells. Analysis methods include: cell count and viability; mycoplasma (1×10 ⁶ cells/average survival concentration;) flow (5×10 ⁶ cells/average survival concentration;) and IFN-g assay (5×10 ⁶ cells - 1 x ¹⁰⁶ cells; IFN-γ assay requires 8-10 x ¹⁰⁶ cells.

Calculate the volume of the cell fraction cryopreserved at 10 x ¹⁰⁶ cells/ml and calculate the number of vials to prepare. Day 16-17 _

Wash buffer preparation (1% HSA Plasmalyte A). Transfer HSA and Plasmalyte to 5 L bags to prepare LOVO Wash Buffer. Transfer a total volume of 125 mL of 25% HSA to a 5 L bag using aseptic conditions. Remove 10 mL or 40 mL of wash buffer and transfer to "IL-2 6×10 ⁴ IU/mL tube" (10 mL if pre-prepared IL-2, or 10 mL if freshly prepared IL-2 40 mL).

Calculate the volume of reconstituted IL-2 added to Plasmalyte + 1% HSA: Volume of reconstituted IL-2 = (final concentration of IL-2 x final volume)/specific activity of IL-2 (based on standard assay Law). The final concentration of IL-2 was 6×10 ⁴ IU/mL. The final volume is 40 mL.

Take the calculated initial volume of IL-2 needed to reconstitute IL-2, and transfer to an "IL-2 6 x 10 ⁴ IU/mL" tube. 100 μL of IL-2 6×10 ⁶ IU/mL from a pre-prepared aliquot was added to a tube labeled “IL-2 6×10 ⁴ IU/mL” containing 10 mL of LOVO wash buffer.

Remove about 4500 mL of supernatant from the G-REX-500MCS culture flask. Swirl the remaining supernatant and transfer the cells to a cell collection bag. Repeat for all G-REX-500MCS flasks.

60 mL of supernatant was removed and added to supernatant tubes for quality control analysis, including mycoplasma detection. Store at +2-8°C.

Cell collection. Count the cells. Prepare four 15 mL conical tubes with 4.5 mL of AIM-V. Such tubes can be prepared in advance. Optimum range = between 5 x 10 ⁴ and 5 x 10 ⁶ cells/ml. (1:10 dilution recommended). For a 1:10 dilution, add 500 µL of CF to 4500 µL of AIM V previously prepared. Record the dilution factor. Calculate pre-LOVO (survival + dead) TC (total cells) = average total cell concentration (pre-LOVO TC concentration) (live + dead) X source bag volume Calculate TVC (total viable cells) before LOVO (live) = average Total viable cell concentration (viable cells). Concentration (TVC before LOVO) (live) X volume of LOVO source bag

When the number of total cells (TC) was >5×10 ⁹ , 5×10 ⁸ cells were taken out for cryopreservation as MDA reserved samples. 5 x ¹⁰⁸ ÷ average TC concentration (step 14.44) = volume to be removed.

When the total cell (TC) number was ≤5×10 ⁹ , 4×10 ⁶ cells were removed for cryopreservation as MDA reserve samples. 4 x 10 ⁶ ÷ total TC concentration = volume to be removed.

When determining the total cell number, the number of cells to be removed should allow 150 x ¹⁰⁹ viable cells to remain. Confirm pre-LOVO TVC 5×10 ⁸ or 4×10 ⁶ or not applicable. Calculate the volume of cells removed from the LOVO source bag.

Count the total number of remaining cells remaining in the bag. TC (total cells) before LOVO were calculated. [Average total cell concentration × remaining volume = TC pre-LOVO remaining]

According to the total number of remaining cells, select the corresponding process in Table 41.

Select the amount of IL-2 added corresponding to the process used. The volume is calculated as: retentate volume x 2 x 300 IU/mL = IU of IL-2 required. Required IU of IL-2/6×10 ⁴ IU/mL=the volume of IL-2 added after the LOVO bag. Record all volumes added. Samples were obtained in frozen vials for further analysis.

The cell product was mixed thoroughly. All bags were sealed for further processing, including cryopreservation as appropriate.

Perform endotoxin, IFN-γ, sterility, and other analyzes on obtained frozen vial samples as required. Example 9 : GEN 2 and GEN 3 Exemplary Procedures

This example shows Gen 2 and Gen 3 programs. Programs Gen 2 and Gen 3 TILs generally consist of autologous TILs obtained from individual patients by surgically resecting the tumor and then expanding ex vivo. The initial first expansion step of the Gen 3 program was cell culture in the presence of interleukin-2 (IL-2) and the monoclonal antibody OKT3, which targets irradiated peripheral blood mononuclear cells (PBMC) T cell co-receptor CD3 on the backbone.

The manufacture of Gen 2 TIL products consists of two stages: 1) Pre-rapid amplification (Pre-REP), and 2) Rapid amplification protocol (REP). During Pre-REP, resected tumors were cut into ≤50 fragments each 2-3 mm in size, which were treated with serum-containing medium (RPMI 1640 medium supplemented with 10% HuSAB) and 6,000 IU/mL Interleukin-2 (IL-2) was cultured for a period of 11 days. On day 11, TILs were harvested and introduced into a large-scale secondary REP expansion. REP consisted of: activation of ≤200× ¹⁰⁶ feeder cells from pre-pregnancy in a co-culture of 5× ¹⁰⁹ irradiated allogeneic PBMC feeder cells in a volume of 5 L of CM2 supplemented with 3000 IU/mL rhIL-2. Live cells of REP were maintained for 5 days, and the feeder cells were loaded with 150 µg of monoclonal anti-CD3 antibody (OKT3). On day 16, the culture volume was reduced by 90% and the cell fraction was split into multiple G-REX-500 flasks at ≥ 1 x ¹⁰⁹ viable lymphocytes/flask and made up to 5 L with CM4. TILs were incubated for an additional 6 days. REPs were collected on day 22, washed, reconstituted and stored frozen before being shipped to the clinical site at -150°C for infusion.

The manufacture of Gen 3 TIL products consists of three phases: 1) an initial first expansion protocol; 2) a rapid second expansion protocol (also known as the Rapid Expansion Phase or REP); and 3) subculture analysis. bottle. To achieve initial first expansion TIL propagation, resected tumors were dissected into < 120 fragments each 2-3 mm in size. On day 0 of initiation of the first expansion, a feeder of approximately 2.5 x ¹⁰⁸ allogeneically irradiated PBMC feeder cells was established on a surface area of approximately 100 cm in ^each of the three 100 MCS containers layer, the feeder cells are loaded with OKT-3. Tumor fragments were distributed among three 100 MCS containers and cultured for a period of 7 days, each container containing 500 mL of serum-containing CM1 medium with 6,000 IU/mL interleukin-2 (IL-2) and 15 μg OKT-3. On day 7, REP was initiated by pooling an additional feeder layer of approximately 5 x ¹⁰ allogeneic irradiated PBMC feeder cells loaded with OKT-3 into each of three 100 MCS containers In the stage of tumor fragmentation culture, cultured with 500 mL CM2 medium, 6,000 IU/mL IL-2 and 30 µg OKT-3. REP initiation was enhanced by activating the entire initial first expansion culture in the same vessel by transferring OKT3-loaded feeder cells into a 100 MCS vessel using closed system fluid. For Gen 3, TIL scale-up or splitting involved the following procedural steps: Scaling the entire cell culture via closed system fluid transfer to larger vessels and transferring (from 100 M flasks to 500 M flasks) and Add an additional 4 L of CM4 medium. REPs were collected on day 16, washed, reconstituted and stored frozen before being shipped to the clinical site at -150°C for infusion.

Overall, the Gen 3 process is a shorter, more scalable, and easily modifiable amplification platform that will accommodate robust manufacturing and program comparability.

On day 0, for both procedures, tumors were washed 3 times and fragments were randomized and divided into two pools; one pool for each procedure. For the Gen 2 program, transfer fragments to a GREX 100MCS flask with 1 L of CM1 medium containing 6,000 IU/mL rhIL-2. For the Gen 3 program, fragments were transferred to a G-REX-100MCS flask with 500 mL CM1 containing 6,000 IU/mL rhIL-2, 15 μg OKT-3, and 2.5 x ¹⁰⁸ feeder cells. Inoculation of TILs on the start day of Rep was carried out on different days according to each program. For the Gen 2 program, in which the volume of the G-REX-100MCS flask was reduced by 90%, the collected cell suspension was transferred to a new G-REX-500MCS, and on day 11 in the presence of IL-2 (3000 IU/mL ) and start REP in CM2 medium with 5×10 ⁹ feeder cells and OKT-3 (30 ng/mL). According to the protocol, cells were expanded and split on day 16 into multiple G-REX-500 MCS flasks with CM4 medium and IL-2 (3000 IU/mL). Then, according to the protocol, cultures were harvested on day 22 and stored frozen. For the Gen 3 program, REP initiation occurred on day 7, where the same G-REX-100MCS was used for REP initiation. Briefly, 500 mL of CM2 medium containing IL-2 (6000 IU/mL) and 5×10 ⁸ feeder cells and 30 μg OKT-3 was added to each flask. On days 9-11, cultures were scaled up longitudinally. The entire volume of G-REX100M (1 L) was transferred to G-REX-500MCS, and 4 L of CM4 containing IL-2 (3000 IU/mL) was added. The flasks were incubated for 5 days. Cultures were harvested on day 16 and stored frozen.

Three different tumors were included in the comparison, two lung tumors (L4054 and L4055) and one melanoma (M1085T).

For L4054 and L4055, CM1 (medium 1), CM2 (medium 2) and CM4 (medium 4) media were prepared in advance and kept at 4°C. CM1 and CM2 media were prepared without filtration to compare cell growth with and without media filtration.

For L4055 tumors, media was warmed at 37°C for up to 24 hours at REP initiation and longitudinal expansion in size.

result. Gen 3 results were within 30% of Gen 2 results for total viable cells achieved. After restimulation, the Gen 3 final product exhibited higher IFN-γ production. The Gen 3 final product exhibited increased diversity of the colonies as measured by the total unique CDR3 sequences present. Gen 3 end products exhibited longer average telomere lengths.

Pre-REP and REP amplification for Gen 2 and Gen 3 procedures followed the procedures described above. For each tumor, both pools contained an equal number of fragments. Due to the small size of the tumors, the maximum number of fragments per flask could not be achieved. Total pre-REP cells (TVCs) were collected and counted on day 11 of the Gen 2 program and on day 7 of the Gen 3 program. To compare the two pre-REP groups, the cell count was divided by the number of fragments presented in culture to calculate the mean of viable cells per fragment. As indicated in Table 51 below, more cells were consistently grown per fragment in the Gen 2 program compared to the Gen 3 program. The number of TVCs expected for the Gen 3 program on day 11 was extrapolated, and the number of TVCs was calculated by dividing the pre-REP TVC by 7 and then multiplying by 11.

For Gen 2 and Gen 3 programs, TVCs were counted according to the program conditions and the percent viable cells were generated on each day of the program. At harvest, day 22 (Gen 2) and day 16 (Gen 3) cells were harvested and TVC counts determined. Next, the TVC was divided by the number of fragments presented on day 0 to calculate the average number of viable cells per fragment. Fold amplification was calculated by dividing the collected TVC by the REP starting TVC. As shown in Table 52, comparing Gen 2 and Gen 3, for L4054, the fold amplification is similar; in the case of L4055, the fold amplification of the Gen 2 process is higher. Specifically, in this case, the medium was warmed 24 hours prior to the day of REP initiation. Higher fold amplification was also observed in Gen 3 for M1085T. The number of TVCs expected for the Gen 3 program on Day 22 was extrapolated and calculated by dividing the REP TVC by 16 and then multiplying by 22.

Table 53: Percent Survival of TIL Final Products: After collection, the final TIL REP products were compared against release criteria for percent viable. All criteria for Gen 2 and Gen 3 programs exceeded the 70% survival criterion and were comparable across programs and tumors.

After collection, the final TIL REP product was compared against the release criterion of percent survival. All criteria for Gen 2 and Gen 3 programs exceeded the 70% survival criterion and were comparable across programs and tumors.

As the number of fragments per flask was lower than the maximum required number, an estimated cell count was calculated for each tumor on the day of collection. This estimate was based on the expectation that clinical tumors would be large enough to inoculate 2 or 3 culture flasks on day 0.

Immunophenotype - Comparison of phenotypic markers of TIL end products. The three tumors L4054, L4055 and M1085T all underwent TIL expansion during Gen 2 and Gen 3. After harvesting, REP TIL final products were subjected to flow cytometric analysis to test for purity, differentiation and memory markers. The percentage of TCR a/b+ cells was over 90% for all conditions.

TILs collected from the Gen 3 program showed higher expression levels of CD8 and CD28 compared to TILs collected from the Gen 2 program. Gen 2 programs showed higher CD4+ percentages.

TILs collected from the Gen 3 program showed higher central memory compartment representation compared to TILs collected from the Gen 2 program.

Activation and depletion markers were analyzed in TILs from two tumors, L4054 and L4055, to compare the final TIL products from the Gen 2 and Gen 3 TIL expansion procedures. Activation and depletion markers are similar for Gen 2 and Gen 3 processes.

Interferon gamma secretion after restimulation. For L4054 and L4055, on the day of collection, day 22 for Gen 2 and day 16 for Gen 3, TILs were restimulated overnight using anti-CD3 discs coated. Restimulation of M1085T was performed using anti-CD3, CD28 and CD137 beads. Supernatants were harvested after 24 hours restimulation under all conditions and frozen. Supernatants from both procedures were assessed for IFNy analysis by ELISA at the same time using the same ELISA plate. Higher production of IFNγ from the Gen 3 program was observed in the three tumors analyzed.

Measurement of IL-2 content in culture medium. To compare IL-2 depletion of the Gen 2 and Gen 3 programs, cell supernatants were collected from tumors L4054 and L4055 at REP initiation, scale-up and collection days. The amount of IL-2 in the cell culture supernatant was measured by the Quantitate ELISA kit from R&D. The overall trend indicated that higher IL-2 concentrations were maintained in the Gen 3 program when compared to the Gen 2 program. This may be attributed to the higher concentration of IL-2 (6000 IU/mL) at the start of REP in Gen 3 and the residual culture medium throughout the procedure.

Metabolic substrate and metabolite analysis. The content of metabolic substrates such as D-glucose and L-glutamine was measured as a proxy for total media consumption. Measure its reverse metabolites, such as lactate and ammonia. Glucose is a monosaccharide in the medium that mitochondria use to generate energy in the form of ATP. When glucose is oxidized, lactic acid (lactate of lactate) is produced. Large amounts of lactate are produced during exponential cell growth. Higher lactate content can negatively affect cell culture procedures.

Spent medium for L4054 and L4055 was collected at REP initiation, scale-up and collection days of the Gen 2 and Gen 3 programs. Spent medium was collected on days 11, 16 and 22 of Gen 2 and on days 7, 11 and 16 of Gen 3. The concentrations of glucose, lactate, glutamine, GlutaMax™ and ammonia in the supernatant were analyzed on a CEDEX bioanalyzer.

An unstable essential amino acid required in L-glutamine-based cell culture medium formulations. Glutamine contains amines, and this amide structural group transports and delivers nitrogen to cells. When L-glutamine oxidizes, cells produce toxic ammonia by-products. To counteract the degradation of L-glutamine, the media of Gen 2 and Gen 3 programs are supplemented with GlutaMax™, which is more stable in aqueous solution and does not spontaneously degrade. In both tumor lines, the Gen 3 group showed a decrease in L-glutamine and GlutaMax™ and an increase in ammonia throughout the REP during this procedure. In the Gen 2 group, constant L-glutamine and GlutaMax™ concentrations, and a slight increase in ammonia production were observed. Ammonia for the Gen 2 and Gen 3 procedures were comparable on the day of collection and showed minor differences in L-glutamine degradation.

Telomeric repeats were measured by Flow-FISH. The average length of telomeric repeats on L4054 and L4055 in the Gen 2 and Gen 3 programs was measured using Flow-FISH technology. Measurements of relative telomere length (RTL) were calculated using the Telomere PNA Kit/FITC for Flow Cytometry Analysis from DAKO. Gen 3 showed comparable telomere lengths to Gen 2.

CD3 analysis. To determine the strain diversity of the cell products produced in each procedure, the collected TIL end products of L4054 and L4055 were sampled and the strain diversity was determined by sequencing the T cell receptor CDR3 portion. analyze.

Table 55 shows a comparison of the percentage of unique CDR3 sequences on consensus L4054 on TIL harvested cell products between Gen 2 and Gen 3. There were 199 sequences shared between the Gen 3 and Gen 2 final products, and 97.07% of the unique CDR3 sequences corresponding to the top 80% of the Gen 2 final products were shared with the Gen 3 final products.

Table 56 shows a comparison of the percentage of unique CDR3 sequences on consensus L4055 on TIL harvested cell products between Gen 2 and Gen 3. There were 1833 sequences shared between the Gen 3 and Gen 2 final products, and 99.45% of the unique CDR3 sequences corresponding to the top 80% of the Gen 2 final products were shared with the Gen 3 final products.

CM1 and CM2 media were pre-prepared unfiltered and kept at 4°C until use of tumor L4055 for Gen 2 and Gen 3 procedures.

For L4055 tumors, media was warmed at 37°C for 24 hours on REP initiation day for Gen 2 and Gen 3 procedures.

LDH was not measurable in the supernatant collected during the procedure.

Perform M1085T TIL cell counts with a K2 cellometer.

Samples such as supernatants for metabolic analysis, TIL products for activation and depletion marker analysis, telomere length and CD3-TCR vb analysis were not available on tumor M1085T.

in conclusion. This example compares 3 independent donor tumor tissues for functional quality attributes and extended phenotypic characterization and media consumption for the Gen 2 and Gen 3 programs.

Gen 2 and Gen 3 pre-REP and REP expansion comparisons were assessed in terms of total viable cell numbers generated and viability of the total nucleated cell population. There was no comparison between Gen 2 (22 days) and Gen 3 (16 days) TVC cell doses on the day of collection. Gen 3 cell dose was lower than Gen 2 and was approximately 40% of the total viable cell number collected at the time of collection.

Gen 3 program extrapolated cell numbers were calculated assuming pre-REP collection on day 11 instead of day 7 and REP collection on day 22 instead of day 16. In both cases, Gen 3 showed a closer TVC number compared to the Gen 2 program, suggesting that early activation enhances TIL growth.

In the case of extrapolating the value of extra flasks (2 or 3) in the Gen 3 program, it was assumed that the size of the tumors treated was larger and the maximum number of fragments required for each program as described was achieved. It was observed that TVCs collected on day 16 of the Gen 3 procedure achieved a similar dose as compared to that of the Gen 2 procedure on day 22. This observation is important and indicates that early activation of the culture shortens TIL treatment time.

Gen 2 and Gen 3 pre-REP and REP expansion comparisons were assessed in terms of total viable cell numbers generated and viability of the total nucleated cell population. There was no comparison between Gen 2 (22 days) and Gen 3 (16 days) TVC cell doses on the day of collection. Gen 3 cell dose was lower than Gen 2 and was approximately 40% of the total viable cell number collected at the time of collection.

In terms of phenotypic characterization, three tumors were observed to have higher expression levels of CD8+ and CD28+ in the Gen 3 program compared to the Gen 2 program.

Gen 3 programs showed slightly higher central memory compartments compared to Gen 2 programs.

Despite the shorter duration of the Gen 3 program, the Gen 2 and Gen 3 programs showed comparable activation and depletion markers.

IFN gamma (IFN gamma) production of the Gen 3 end product was 3-fold higher than Gen 2 in the three tumors analyzed. This data suggests that the Gen 3 program produces a powerful and more potent TIL product compared to the Gen 2 program, which may be due to the higher expression of CD8 and CD28 in Gen 3. Phenotype characterization showed that compared with the Gen 2 program, the CD8+ and CD28+ expression levels of Gen 3 on the three tumors showed a positive trend.

Gen 2 and Gen 3 TIL end products have comparable telomere lengths.

The glucose and lactate contents of the Gen 2 and Gen 3 final products were comparable, indicating that the nutrient content of the medium in the Gen 3 procedure was not affected, as no volume reduction was performed on each day of the procedure compared to Gen 2. Small removal and overall medium volumes in this procedure are small.

Compared to the Gen 2 process, the processing time of the entire Gen 3 process is reduced by about two times, which will significantly reduce the cost of goods (COG) of TIL products amplified by the Gen 3 process.

IL-2 depletion showed a general trend of IL-2 depletion in the Gen 2 program and was higher in the Gen 3 program since the old medium was not removed.

The Gen 3 program showed a high diversity of colonies as determined by CDR3 TCRab sequence analysis.

Addition of feeder cells and OKT-3 at pre-REP day 0 allowed early activation of TILs and allowed TIL growth using the Gen 3 program.

實例 10 ：例示性 GEN 3 過程 ( 亦稱為 GEN 3.1) Table 57 describes various embodiments and results of the Gen 3 process compared to the current Gen 2 process.

Example 10 : Exemplary GEN 3 Process ( also known as GEN 3.1)

This example describes other studies on "Comparability between Gen 2 and Gen 3 programs for TIL expansion". The Gen 3 program was modified to include an activation step early in the program with the aim of increasing the final total viable cell (TVC) output while maintaining phenotypic and functional profiles. As described below, the Gen 3 embodiment was modified into another embodiment and referred to herein as Gen 3.1 in this example.

In some embodiments, the Gen 3.1 TIL manufacturing procedure has four operator interventions: 1. Tumor Fragment Isolation and Activation: On Day 0 of the procedure, tumors are segmented and final fragments each approximately 3 x 3 mm (total up to 240 fragments) and cultured in 1-4 G-REX100MCS flasks. Each flask contained up to 60 fragments, 500 mL CM1 or DM1 medium supplemented with 6,000 IU rhIL-2, 15 μg OKT3, and 2.5 x ¹⁰⁸ irradiated allogeneic monocytes. Cultures were grown at 37°C for 6-8 days. 2. Reactivation of TIL cultures: On days 7-8, in both cases, cultures were supplemented by slow addition of 6,000 IU rhIL-2, 30 μg OKT3, and 5×10 ⁸ irradiated allogeneic mononuclear CM2 or DM1 medium for cells was supplemented. Be careful not to damage existing cells at the bottom of the flask. Cultures were incubated at 37°C for 3-4 days. 3. Vertical expansion of the culture scale: carried out on the 10th-11th day. During vertical scale-up of cultures, in both cases the entire contents of G-REX100MCS were transferred to G-REX500MCS flasks containing 4 L of CM4 or DM2 supplemented with 3,000 IU/mL IL-2. Flasks were incubated at 37°C for 5-6 days until harvested. 4. Harvesting/Washing/Preparation: On day 16-17, the flasks were reduced in volume and pooled. Cells were concentrated and washed with PlasmaLyte A pH 7.4 containing 1% HSA. The washed cell suspension was mixed with CryoStor10 at a ratio of 1:1, and rhIL-2 was supplemented to reach a final concentration of 300 IU/mL.

DP was cryopreserved by controlled rate freezing and stored in gas phase liquid nitrogen. **Complete Standard TIL Medium 1, 2, or 4 (CM1, CM2, CM4) can be substituted for CTS™ OpTmizer™ T Cell Serum-Free Expansion Medium, called Synthetic Medium (DM1 or DM2), as described above.

Program description. On day 0, tumors were washed 3 times and then fragmented into 3x3x3 final fragments. After whole tumors were fragmented, the final fragments were then equally randomized and divided into three pools. A pool of randomized fragments was introduced into each group, with equal numbers of fragments added according to the three experimental matrices.

Throughout the TIL expansion procedure, tumor L4063 expansion was performed using standard media and tumor L4064 expansion was performed using synthetic media (CTS OpTmizer). The components of the medium are described herein.

CM1 Complete Medium 1: RPMI+Glutamine supplemented with 2 mM GlutaMax™, 10% Human AB Serum, Gentamycin (50 μg/mL), 2-Mercaptoethanol (55 μM). The final media formulation was supplemented with 6000 IU/mL IL-2.

CM2 complete medium 2: 50% CM1 medium + 50% AIM-V medium. The final media formulation was supplemented with 6000 IU/mL IL-2.

CM4 Complete Medium 4: AIM-V supplemented with GlutaMax™ (2 mM). The final media formulation was supplemented with 3000 IU/mL IL-2.

CTS OpTmizer CTS™ OpTmizer™ T Cell Expansion Basal Medium is supplemented with CTS™ OpTmizer™ T Cell Expansion Supplement (26 mL/L).

DM1: CTS™ OpTmizer™ T Cell Expansion Basal Medium supplemented with CTS™ OpTmizer™ T Cell Expansion Supplement (26 mL/L), CTS™ Immune Cell SR (3%) and GlutaMax™ (2 mM). The final formulation was supplemented with 6,000 IU/mL IL-2.

DM2: CTS™ OpTmizer™ T Cell Expansion Basal Medium supplemented with CTS™ OpTmizer™ T Cell Expansion Supplement (26 mL/L) and CTS™ Immune Cell SR (3%) and GlutaMax™ (2 mM). The final formulation was supplemented with 3,000 IU/mL IL-2.

All types of media used, i.e., complete (CM) and defined (DM) media, were prepared in advance, kept at 4°C until the day before use, and pre-warmed in an incubator at 37°C prior to the day of treatment Up to 24 hours.

Reactivation of TIL cultures of both tumors was performed on day 7. L4063 was scaled up on day 10 and L4064 on day 11. Both cultures were harvested on day 16 and stored frozen.

The result obtained. Cell counts and percent viability were determined for Gen 3.0 and Gen 3.1 procedures. Amplification under all conditions followed the details described in this example.

For each tumor, fragments were divided into three pools of equal number. Due to the small size of the tumors, the maximum number of fragments per flask could not be achieved. For three different procedures, the total number of viable cells and cell viability under each condition were assessed. Cell counts were determined for TVCs at day 7 for reactivation, for TVCs at day 10 (L4064) or at day 11 (L4063) for scale-up, and for TVCs harvested at day 16/17.

Cell counts on days 7 and 10/11 were considered FIO. Fold amplification was calculated by dividing the day 16/17 collection day TVC by the day 7 reactivation day TVC. To compare the three groups, the TVC on the day of collection was divided by the number of fragments added to the culture at day 0 in order to calculate the mean value of viable cells per fragment.

Cell count and viability analysis were performed on L4063 and L4064. Gen 3.1—For both tumors, the test program produced more cells per fragment than the Gen 3.0 program.

Total viable cell count and expansion fold; % survival during the procedure. Percent survival under all conditions was obtained after reactivation, scale-up and harvesting. At day 16/17 collection, the final TIL was compared against the release criteria of % survival. All conditions exceeded the 70% survival criterion and were comparable across procedures and tumors.

Immunophenotyping - Phenotypic characterization of TIL end products. Flow cytometry analysis was performed on the final product to test for purity, differentiation and memory markers. The population percentages of TCRα/β, CD4+ and CD8+ cells were constant under all conditions.

Perform extended phenotyping of REP TIL. For both tumors, TIL products showed a higher percentage of CD4+ cells under Gen 3.1 conditions compared to Gen 3.0, and a higher percentage of CD4+ cells from Gen 3.0 compared to Gen 3.1 conditions in both conditions. Percentage of CD28+ cells of the CD8+ population.

TILs collected from the Gen 3.0 and Gen 3.1 programs showed comparable phenotypic markers, such as CD27 and CD56 expression on CD4+ and CD8+ cells, and comparable CD28 expression on CD4+ circled cell populations. Comparison of memory markers for TIL end products:

Frozen samples of TIL collected on day 16 were stained for analysis. The TIL memory states of Gen 3.0 and Gen 3.1 programs are comparable. Activation and depletion marker comparison for TIL end products:

Activation and depletion markers for the Gen 3.0 and Gen 3.1 programs for CD4+ and CD8+ cells were comparable.

Interferon gamma secretion after restimulation. For L4063 and L4064, the collected TILs were restimulated overnight using anti-CD3 coated discs. Higher IFNγ production from the Gen 3.1 program was observed in both tumors analyzed compared to the Gen 3.0 program.

Measurement of IL-2 content in culture medium. To compare IL-2 consumption between all conditions and procedures, reactivation initiation at day 7, scale-up at day 10 (L4064)/day 11 (L4063) and day of collection at day 16/day 17 Cell supernatants were collected and frozen. Subsequently, the supernatant was thawed and then analyzed. The amount of IL-2 in cell culture supernatants was measured by the manufacturer's protocol.

IL-2 depletion was comparable throughout the Gen 3 and Gen 3.1 programs over the entire program period assessed under the same media conditions. The collected spent media of L4063 and L4064 were analyzed for IL-2 concentration (pg/mL).

Metabolite analysis. For each condition, spent cells were collected from L4063 and L4064 at Day 7 reactivation initiation, Day 10 (L4064)/Day 11 (L4063) scale-up, and Day 16/Day 17 collection days. culture supernatant. The concentrations of glucose, lactate, glutamine, GlutaMax™ and ammonia in the supernatant were analyzed with a CEDEX bioanalyzer.

Compared with the complete medium (2 g/L), the glucose concentration in the synthetic medium was higher at 4.5 g/L. Overall, the concentration and consumption of glucose in the Gen 3.0 and Gen 3.1 programs were comparable among the media types.

An increase in lactate was observed and was comparable between Gen 3.0 and Gen 3.1 conditions and between the two media (complete and synthetic) used for reactivation expansion.

In some cases, standard basal medium contained 2 mM L-glutamine and was supplemented with 2 mM GlutaMax™ to compensate for the natural degradation of L-glutamine to L-glutamine and ammonia under culture conditions.

In some cases, the synthetic (serum-free) medium used was L-glutamine-free compared to basal medium and was only supplemented with GlutaMax™ at a final concentration of 2 mM. GlutaMax™ is a dipeptide containing L-alanine and L-glutamine, which is more stable than L-glutamine in aqueous solution and will not spontaneously degrade into glutamic acid and ammonia. In fact, the dipeptide gradually dissociates into individual amino acids, thereby maintaining a low but sufficient concentration of L-glutamine to maintain robust cell growth.

In some cases, concentrations of glutamine and GlutaMax™ decreased slightly on the scale-up day, but appeared to increase to similar or closer levels on the harvest day compared to the reactivation day. For L4064, glutamine and GlutaMax™ concentrations showed a slight decrease at similar rates between conditions throughout the procedure.

Ammonia concentrations were higher in samples grown in standard media containing 2 mM Glutamine + 2 mM GlutaMax™ than in samples grown in synthetic media containing 2 mM GlutaMax™. Furthermore, as expected, ammonia gradually increased or accumulated during the incubation. Under the three different test conditions, there was no difference in ammonia concentration.

Telomere repeats were determined by Flow-FISH. The average length of telomeric repeats at L4063 and L4064 in the Gen 3 and Gen 3.1 programs was measured using Flow-FISH technology. Measurements of relative telomere length (RTL) were calculated using the Telomere PNA Kit/FITC for Flow Cytometry Analysis from DAKO. Perform telomere analysis. Telomere length in the samples was compared to a control cell line (1301 leukemia). The control cell line is a tetraploid cell line with long stable telomeres, which allows calculation of relative telomere length. The Gen 3 and Gen 3.1 programs assessed in both tumors showed comparable telomere lengths.

TCR Vβ lineage analysis

To determine the colony diversity of the cellular products produced in each procedure, TIL final products were analyzed by sequencing the CDR3 portion of the T cell receptor for colony diversity analysis.

Compare three parameters between three conditions: ● Diversity index of unique CDR3 (uCDR3) ● Total uCDR3 percentage ● For the top 80% of uCDR3: o Comparing the percentage of shared uCDR3 copies o Compare frequencies of unique pedigrees

Control and Gen 3.1 test, the percentage of unique CDR3 sequences shared by TIL collected cell products: Gen 3 and Gen 3.1 test final products share 975 sequences, which is equivalent to 88% of the unique CDR3 sequences before Gen 3 and Gen 3.1. .

Control and Gen 3.1 test, the percentage of unique CDR3 sequences shared by TIL collected cell products: Gen 3 and Gen 3.1 test final products share 2163 sequences, which is equivalent to 87% of the unique CDR3 sequences before 80% of Gen 3 and Gen 3.1 .

The number of unique CD3 sequences was identified from 1 x ¹⁰⁶ cells collected at day 16 of the different procedures. The Gen 3.1 test conditions showed slightly higher diversity of colonies compared to Gen 3.0 based on the number of unique peptide CDRs within the sample.

The Shannon entropy diversity index is a reliable and commonly used comparative measure, as the Gen 3.1 condition of both tumors showed slightly higher diversity than the Gen 3 program, indicating that the Gen 3.1 test condition had a higher TCRVβ repertoire than the Gen 3.0 The procedure is more polygenic.

Furthermore, for tumors L4063 and L4064, the TCR Vβ repertoire of the Gen 3.1 test condition showed over 87% overlap with the corresponding repertoire of the Gen 3.0 program.

On the day of reactivation, the IL-2 concentration values of the spent medium used for the Gen 3.1 test L4064 were lower than expected (similar to the Gen 3.1 control and Gen 3.0 conditions).

This low value could be attributed to pipetting error, but it was not possible to repeat the analysis due to the small number of samples collected.

in conclusion. Gen 3.1 test conditions (including feeder cells at day 0 and OKT-3) showed a higher TVC for the cell dose at day 16 harvest compared to Gen 3.0 and Gen 3.1 controls. Under Gen 3.1 test conditions, the TVC of the final product is about 2.5 times higher than that of Gen 3.0.

For both tumor samples tested, the Gen 3.1 test condition supplemented with OKT-3 and feeder cells at day 0 reached the maximum capacity of the culture flask at the time of collection. Under these conditions, the final cell dose can be between 80-100 x ¹⁰⁹ TILs if starting up to 4 culture flasks on day 0.

All quality attributes such as phenotypic characterization including purity, depletion, activation and memory markers of the final TIL product were maintained between the Gen 3.1 assay and the Gen 3.0 program.

In both tumors analyzed, IFN-γ production of final TIL products was 3-fold higher in Gen 3.1 supplemented with feeder cells and OKT-3 at day 0 than in Gen 3.0, indicating that the Gen 3.1 procedure produces potent TIL products.

No differences in glucose or lactate content were observed under each test condition. No differences in glutamine and ammonia were observed between the Gen 3.0 and Gen 3.1 procedures under various media conditions. The lower glutamine content in the medium did not limit cell growth and indicated that the addition of GlutaMax™ to the medium alone was sufficient to provide the nutrients needed for cell proliferation.

Scale-up was performed on day 11 and day 10, respectively, and showed no significant difference in the number of cells reached on the harvest day of the procedure, and metabolite consumption was comparable throughout the procedure in both cases. This observation indicates that Gen 3.0 optimization procedures can be flexible in the number of processing days, thereby facilitating flexibility in manufacturing schedules.

The Gen 3.1 program with the addition of feeder cells and OKT-3 at day 0 showed higher colony diversity as measured by CDR3 TCRab sequence analysis compared to Gen 3.0.

Figure 32 depicts an example of a Gen 3 program (Gen 3 optimized program). Gen 3 Optimizer TIL expansion can be performed using standard media as well as CTS Optimizer serum-free media. In the case of CTS Optimizer, serum-free medium is recommended to increase the final concentration of GlutaMax™ in the medium to 4 mM. Example 15 : Method Tumor Preparation for TIL Expansion in Infiltrating Lymphocytes (TIL) Associated with CD39/CD69 Selection and Gene Knockout

Freshly resected tumor samples were used for digestion, sorting and Gen 2 product generation. Photographs were taken and tumors were removed from the packaging, vials were weighed in the presence and absence of tumors and tumor masses calculated.

Fragment the whole tumor into approximately 4-6 mm ^fragments for tumor digestion. Tumors were digested and expanded using the Gen 2 protocol, depending on the cell population used for expansion. Enzymes for tumor digestion ( using research grade DNase , collagenase and hyaluronidase )

Lyophilized enzymes were reconstituted with the amount of sterile HBSS indicated below for each digestive enzyme. Prepare these enzymes at 10X. Pipette up and down several times and swirl to ensure complete recovery.

1 g collagenase IV (Sigma, MO, C5138) was reconstituted in 10 ml HBSS (to yield a 100 mg/mL stock solution). Mix by pipetting up and down to dissolve. If not dissolved after reconstitution, place in 37°C H2O bath for 5 minutes. Aliquot into 1 ml vials. This is a 100 mg/mL 10X working stock solution for collagenase.

A DNase (Sigma, MO, D5025) stock solution (10,000 IU/mL) was prepared. The units of DNase for each batch are provided in the accompanying data sheet. Calculate the volume of HBSS suitable for reconstitution of 100 mg lyophilized DNase stock solution. For example, if the DNase stock solution is 2000 U/mg, the total DNase in the stock solution is 200,000 IU (2000 IU/mg×100 mg). For the working stock solution diluted to 10,000 IU, add 20 ml HBSS (200,000 IU/20 ml=10,000 U/mL) to 100 mg DNase. Aliquot into 1 ml vials. This is a 10,000 IU/mL 10X working stock solution of DNase.

A hyaluronidase 10 mg/mL (Sigma, MO, H2126) stock solution was prepared. A 500 mg vial was reconstituted with 50 ml HBSS to yield a 10 mg/mL stock solution. Aliquot into 1 ml vials. This is a 10 mg/mL 10X working stock solution of Hyaluronidase.

Dilute stock digestive enzymes to 1X. To prepare a 1X working solution, add 500 ml each of collagenase, DNase and hyaluronidase to 3.5 ml HBSS. Add the digestion mix directly to the C-tube. Enzyme preparation for tumor digestion ( using GMP collagenase and dispase )

Lyophilized enzymes were reconstituted with the amount of sterile HBSS indicated below for each digestive enzyme. Make sure to get any residual powder from the side of the bottle and from the protective foil over the opening of the bottle. Pipette up and down several times and swirl to ensure complete recovery.

Collagenase AF-1 (Nordmark, Sweden, N0003554) was reconstituted in 10 ml sterile HBSS. The concentration of the lyophilized stock enzyme is 2892 PZ U per vial. After reconstitution, the collagenase stock solution was 289.2 PZ U/mL.

Dispase (Nordmark, Sweden, N0003553) was reconstituted in 1 ml sterile HBSS. The concentration of the lyophilized stock enzyme is 175 DMC U per vial. Therefore, after reconstitution, the dispase stock solution is 175 DMC/mL.

DNase I (Roche, Switzerland, 03724751) was reconstituted in 1 ml sterile HBSS. The concentration of freeze-dried stock enzyme is 4KU per vial. Therefore, after reconstitution, the DNase stock solution is 4KU per vial.

Prepare working GMP digestion mix. Add 10.2 µl of Dispase (0.36 DMC U/mL), 21.3 µl of Collagenase AF-1 (1.2 PZ/mL) and 250 µl of DNase I (200 U/mL) into 4.7 ml of sterile HBSS. Place the digestion mix directly into the C-tube. Tumor Treatment and Digestion

If GentleMACS OctoDissociator, transfer tumor fragments in 5 ml digestion mix (in HBSS) indicated above to GentleMACS C-Tube (C-shaped tube) or 50 ml conical tube. Transfer 2-3 fragments (4-6 mm) to each C-tube.

Each C-tube (Miltenyi Biotec, Germany, 130-096-334) was transferred into a GentleMACS OctoDissociator (Miltenyi Biotec, Germany, 130-095-937). Use according to manufacturer's instructions. Note that each tumor histology has a recommended protocol for tumor dissociation. The appropriate program is selected for individual tumor histology. Dissociation will take about one hour.

If the GentleMACS OctoDissociator is not available, a standard spinner is used. 2-3 tumor fragments were placed in 50 ml conical tubes (sealed with parafilm to avoid leakage) and secured to the rotator. Place the spinner in a 37°C, 5% CO2 humidified incubator and keep spinning for 1-2 hours. Alternatively, tumor fragments can be digested overnight at room temperature, also with constant rotation.

After digestion, remove the C-tube from the Octodissociator or rotator. Connect a 0.22 µm filter to sterile Falcon conical tubing. Using a pipette, transfer all contents from a C-tube or 50 ml conical tube (5 ml) through a 0.22 µm filter to a 50 ml conical tube. C-tubes or 50 ml conical tubes were washed with 10 ml HBSS and applied to the strainer. Use the flat end of a sterile syringe plunger to dissociate any remaining undigested tumor through the filter. CM1 or HBSS was added up to 50 ml and the tube was capped.

Samples were pelleted by centrifugation at 1500 rpm (acceleration and deceleration 9) for 5 minutes at room temperature. Carefully remove the liquid and resuspend the pellet in 5 ml CM1 for cell count and viability assessment.

Whole tumor digests were stored for the following: 1. Cell culture (unselected TIL control), 2. FMO flow cytometry control, 3. Presorted whole tumor digest phenotyping analysis, 4. Freeze for tumor reactivity/cell killing assays. The number of cells stored will depend on the total digest yield and tumor histology. Clean up digesta with a debris removal kit

Debris removal solution (Miltenyi Biotec, Germany, Cat. No. 130-109-398) or other equivalent reagents can be used to remove debris from tumor digests according to the manufacturer's instructions.

The tumor cell suspension was centrifuged at 300Xg for 10 minutes at 4°C and the supernatant was aspirated completely.

Carefully resuspend the cell suspension with an appropriate volume of cold buffer according to the table below and transfer the cell suspension to a 15 ml conical tube. Do not vortex. Resuspension (PBS) debris removal solution Covering (PBS) 0.5-1g tissue 6200 µl 1800 µl 4-ml > 0.5 g tissue 3100 µl 900 µl 4-ml

Add an appropriate volume of cold chip removal solution and mix well by pipetting up and down slowly 10-20 times using a 5 ml pipette. Cover very gently with 4 ml of cold buffer. Tilt the tube and pipette very slowly to ensure that the PBS/D-PBS phase covers the cell suspension and that the phases do not mix. The tumor cell suspension was centrifuged at 3000Xg for 10 minutes at full acceleration at 4°C and completely disrupted. Three phases should form. The top two phases were aspirated completely and discarded.

The bottom phase contains debris removal solution and cells. Make sure the bottom volume is at least as large as the debris removal solution added. (ie, if 1 ml of solution is added, at least 1 ml remains at the bottom of the tube).

Make up to 15 ml with cold buffer and invert the tube at least three times. Do not vortex. Centrifugation at 4°C and 1000Xg for 10 minutes will result in complete acceleration and complete disruption. Cells were resuspended in HBSS or medium for cell counting. Stain digested tumors for cell sorting ( ex vivo and post-expansion sorting )

Tumor digests were stained with a cocktail comprising anti-CD69, anti-CD39 and anti-CD3 antibodies according to the following protocol. After counting, cells were resuspended in 10 ml HBSS.

Resuspend the pellet in FACS buffer (1X HBSS, 1 mM EDTA, 2% fetal bovine serum). The amount of FACS buffer added to the pellet was based on the size of the pellet. The staining volume should be about 3 times the size of the pellet. Therefore, if 300 µl of cells are present, the volume of buffer should be at least 900 µl.

For antibody addition, each 100 µl volume is equivalent to one test (antibody titer). That is, if a 1 ml volume is present, a 10-fold amount of titrated antibody is required. Titrated amounts of each of the following antibodies were added per 100 µl volume: anti-CD3-PE-Cy7, anti-CD69 PE and anti-CD39 FITC. Cells were incubated on ice for 30 minutes. Protect from light during cultivation. Stir several times during incubation. Cells were resuspended in 20 ml FACS buffer. Pass the solution through a 70 µm cell strainer into a new 50 ml conical tube. Centrifuge at 400Xg for 5 minutes at room temperature (acceleration and deceleration of 9). Take a suction. Cells were resuspended in FACS buffer containing up to 10e6/mL TOTAL (live+dead). The minimum volume is 300 µl. Transfer to sterile polypropylene FACS tubes or 15 ml conical tubes. 3 ml/tube for FACS sorting. Prepare 15-ml collection tubes for sorted populations. Place 2 ml of FACS buffer in the tube. Cell count and viability

The procedure for obtaining cell and viability counts uses a Nexcelom Cellometer K2 or equivalent cell counter. FACS sorting (FX500 Startup program )

NOTE: Protocols for other flow cytometers will vary and the manufacturer's instructions for the machine are followed and adjusted as necessary.

After priming and when prompted, install all 4 bags of PBS/EDTA buffer. Connect the PEEK sample line. For a detailed description of the procedure, see the LE-FX500 Operator's Guide.

Configure the instrument. When prompted for laser selection, select all 3 lasers. (488nm, 561nm and 638nm). When prompted for filter settings, select the Standard option button. Run automatic calibration. When prompted to load the calibration beads, add 15 drops of the autoset beads to the 5-ml sterile FACS tube. Then follow the prompts.

When prompted to select a setting for automatic calibration, select the Standard option button. While waiting for the calibration to complete prepare the following: • Prepare five sterile 15 ml conical tubes with 10 ml sterile deionized water. • Prepare five sterile 5 ml FACS tubes with 4 ml sterile deionized water. - Prepare five sterile 15 ml conical tubes with 12 ml 70% EtOH. • Prepare five sterile 15 ml conical tubes with 12 ml of 10% sodium hypochlorite.

Cells were sorted according to standard methods used and based on the fluorophore used. sample collection

Verify that the sample and collection chamber are at 5°C and select the stirred sample icon. Click on the Cell Counter tab at the top of the screen and click on the Collect 5°C icon and the Sample 5°C icon. Click on the stir icon. Validation samples are compensated. Click the Compensation tab at the top of the screen.

Place the tube (5 ml FACS tube or 15 ml conical tube) containing the PBMC control on the sample collection platform. Set the sample collection pressure to 6. Start sample collection. Click Circle and Statistics and select 100,000 in the two drop-down menus seen above.

Verify that the cell population is correctly fenced. It may be necessary to adjust the BSC or FSC settings. However, do not adjust the voltage of any other channels.

When finished, adjust circle selection, do not record, click Abort and unload tube. Click on the next tube icon and right click on the tube and select Rename. Label the tube as "PE fmo". Load the tube and press run. NOTE: There are usually not many cells present in this tube. Adjust the circle selection as needed.

When fencing is satisfactory, record as many events as possible (or up to 20,000 CD3 events). The sample pressure can be set to 10 to speed up this collection.

Stop collecting and remove the tube. A previously prepared 15-ml conical tube of sterile dH20 was loaded onto the sample platform. Choose 10 as the sample pressure. Sample collection takes one minute. Repeat until there is no event in the CD3 circle. Remove the dH20 sample tube and discard. Use a permanent marker to draw a line at the bottom and midpoint of the meniscus on the tube to be collected.

Add the sample to be collected to the loading platform. Verify settings. Choose 4 as the sample pressure. Wait for the cells to appear on the screen. About 15 seconds. Pause when fractions are visible. The lower 2 of the 3 fractions were first collected.

The sample chamber door was opened and a 15 ml collection chamber block was loaded into the chamber. Load collection tubes containing collection buffer into the chamber block: Invert the capped tube several times to coat the top of the tube with collection buffer. Tap the tube on the surface of the BSC to remove excess buffer from the top of the tube and cap. CD39-/CD69- (eg, CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative) fractions are selected. The cap is removed and the tube is placed in the sample chamber block. Choose the correct right/left orientation to match the tube position. Click the Load Collection icon.

Adjust the sample pressure so that the total events per second are less than 5,000. Click the Start Sorting icon. The sample pressure was adjusted to maintain a sorting efficiency of at least 85%. 50,000 CD3 events were recorded. Sorting was stopped when the sample was approximately 2/3 empty. Collected samples containing most events were removed. Recap and place on ice or at 4°C. Keeping the sample volume in the collection chamber low allows more cells to be collected during the collection of the highest percentage of cells. Label the collection tube and remove the cap. Place it in the collection chamber.

Select the appropriate left/right orientation for sorting collection. Load collection tube. Press run to record and start sorting. Sorting was stopped when the sample was approximately one-third empty. The collected fractions were removed. Recap and place on ice or at 4°C, and place the CD3 collection tube to the left of the holder.

The left is for CD3 and the right is sorted blank. Continue sorting until all samples are out of the sample tubes. Tubes can be run "dry". Remove the sample tube from the sample chamber and discard. The sorted fractions were removed from the collection chamber. Cap the tube and invert gently several times to incorporate the drop near the top of the tube into the solution. Gently tap the tube on the surface of the BSC to remove excess solution from the top of the tube and cap. Place tubes on ice.

The percent purity of the CD39 ^LO CD69 ^LO fractions was verified. A 14 ml conical tube of sterile dH2O was placed over the sample chamber. Click on the Probe Wash icon. repeat. The dHO tube was removed and the positive fraction tube was added. Change the Sample Pressure value to 10. Click on the next test tube icon. Name the tube with the sample name and "hi select". Click run and record 75 CD3 positive events. Immediately stop the tube and unload it from the sample chamber.

Repeat these steps for the remaining samples. output data

Select the "fmo" tube by double-clicking and save the report. Repeat for each tube collected. REP1 start

Conditions with the least number of cells can be used to determine the number of cells for REP1 initiation. In TIL conditions without selection, the total number of cells in the complete digest required to start REP1 was calculated using the percentage of CD3 cells (determined during sorting), with the same number of CD3 cells as the sorted sample. Total number of complete digest cells used for REP 1 initiation = number of sorted cells seeded in REP 1 /% CD3 cells.

Approximately 1000-100,000 CD3+ cells were mixed with 7 ml or 40 ml CM2 (50% RPMI 1640+10% human serum, Glutamar, Gentamycin and 50% AimV with 3000 IU/mL IL-2, respectively. ) together in a G-REX10 or equivalent bottle for 11 days. At least one G-REX bottle was started for the CD39 ^LO CD69 ^LO sorted population and unselected TILs. Anti-CD3 (clone: OKT3) (30 ng/mL) and feeder cells (1:100 ratio (TIL:feeder cells)) were added to each flask at the initiation of culture.

Cells were grown in plates/flasks for 11 days without media exchange (REP1). For single REP conditions, cells were continued to be cultured for 3-7 days and then characterized.

At the completion of REP1, remove about 30 ml of medium for G-REX 10. Cells were resuspended in residual media by pipetting up and down. Cells were placed in a 50 ml conical tube and centrifuged at 1500 rpm for 5 minutes (acceleration and deceleration of 9).

Media was aspirated and cells were resuspended in 10-20 ml CM2 for enumeration and viability assessment. REP2 start

For mini-REP2 initiation, plate 1e5 cells in G-REX 10 or equivalent with 40 ml CM2 medium and 3000 IU/mL IL-2. Anti-CD3 (clone: OKT3) (30 ng/mL) and feeder cells (1:100 ratio, TIL:feeder cells) were added at the beginning of the culture.

For a "full scale run", expand 2e6-30e6 cells in 1 LCM2 medium with 3000 IU/m IL-2 in G-REX 100M or equivalent. Anti-CD3 (clone: OKT3) (30 ng/mL) and feeder cells (1:100 ratio, TIL:feeder cells) were added at the beginning of the culture.

Media exchange (for micro-scale) or media exchange + splitting (for "full-scale runs") was performed on day 5 of REP2 (day 16 of the process). Reduce the volume of the bottle to about 10 ml (G-REX 10) or 100 ml (G-REX 100M) and supplement to 40 ml (G-REX 10) or 1 L with CM2 or AimV + 3000 IU/mL IL-2 (G-REX 100M). For "full scale runs", the bottles were split 1:2.

On day 11 of REP2 (or day 22 of the process), reduce the volume of the bottle and centrifuge at 1500 rpm for 5 minutes at room temperature (acceleration and deceleration of 9).

Final products were evaluated for cell count, viability, phenotype, interleukin production, TCRVβ lineage analysis, and tumor-specific reactivity.

FIG. 34 provides a schematic of the process outlined in Example 15.

References for Example 15: 1. Rosenberg, SA et al., Durable complete responses in heavily pretreated patients with metastatic melanoma using T cell transfer immunotherapy ( Durable complete responses in heavily preserved patients with metastatic melanoma using T -cell transfer immunotherapy )” . Clinical Cancer Research, 2011. 17 (13): p. 4550-7. 2. Kvistborg, P. et al., " TIL therapy broadens the tumor-reactive CD8 (+) T cell compartment in melanoma patients" . "Tumor Immunology", 2012. 1 (4): pp. 409-418. 3. Simoni, Y. et al., "Bystander CD8(+) T cells are abundant and phenotypically distinct in human tumor infiltrates " . Nature ", 2018. 557 (7706): pp. 575-579. 4. Schumacher, TN and RD Schreiber, " Neoantigens in cancer immunotherapy " . Science, 2015. 348 (6230): pp. 69-74. 5. Turcotte, S. et al., Phenotype and function of T cells infiltrating visceral metastases from gastric cancers and melanoma: implications for adoptive cell transfer therapy )” . Journal of Immunology, 2013. 191 (5): pp. 2217-25. 6. Inozume, T. et al., Selection of CD8+PD-1 + lymphocytes in fresh human melanomas enriched with tumor-reactive T cells for tumor-reactive T cells )” . Journal of Immunology, 2010. 33 (9): pp. 956-64. 7. Gros, A. et al., PD-1 identifies the patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors " . Journal of Clinical Research, 2014. 124 (5): pp. 2246-59. 8. Thommen, DS et al., A transcriptionally and functionally distinct PD-1(+) CD8(+) T cell pool with predictive potential in non-small cell lung cancer treated with PD-1 blockade PD-1(+) CD8(+) T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade )” . National Journal of Medicine, 2018. Example 16 : AKT during Rapid Expansion Protocol (REP) Inhibits Expansion of Memory Characteristics and Interleukin Production of Tumor Infiltrating Lymphocytes (TILs) and Increases Memory Stem Progenitor-Like (CD39 ⁻ CD69 ⁻ ) Cell Population

Patient tumors from different indications were received, fragmented and subjected to an expansion protocol for TIL production. During ex vivo expansion, different doses (0.3 μM and 1 μM) of the pan-AKT inhibitor (AKTi) pataxerti were added to the cultures. The final TIL products were used to assess the expansion potential and phenotypic and functional characteristics of TILs.

AKTi treatment with a dose of 1 μM resulted in equivalent expansion and survival of TILs compared to controls, but doubled the less differentiated CD39-CD69- cell population. This effect was present even after restimulation and these cells displayed reduced expression of CD38 and the transcription factors T-bet and Tox, indicating a less differentiated and depleted phenotype. Importantly, AKTi treatment caused an increased appearance of IFNγ+TNFα+ CD8+ T cells, which translated into increased cytotoxicity.

AKTi treatment during ex vivo TIL expansion increases the proportion of less differentiated, more memory-like functional TILs. Thus, transient inhibition of AKT signaling during TIL expansion may represent an approach for improving TIL quality and enhancing TIL persistence and therapeutic efficacy in the clinical setting. EXAMPLE 17 : AKT Inhibition During Ex vivo TIL Expansion Enhances Interleukin Production and Function While Increasing a Less Differentiated (CD39-CD69-) CD8+ T Cell Population

background

Receiver cell therapy using autologous tumor-infiltrating lymphocytes (TILs) has been demonstrated in metastatic melanoma (Sarnaik AA et al., J Clin Oncology 2021; 39(24):2656-66) and other epithelial malignancies. Durable responses were demonstrated in patients. Recently, a memory stem-progenitor-like (CD39 ^- CD69 ^- ) phenotype was associated with complete regression and TIL persistence in a metastatic melanoma patient cohort (Krishna S et al., Science 2020; 370 (6522) : 1328-34). Strategies for expanding TILs with poorly differentiated and highly stem-like properties can lead to improved persistence, functionality and better antitumor activity.

Pharmacological inhibition of protein kinase B (AKT) in TILs has been shown to induce transcriptional, metabolic and functional properties characteristic of memory T cells (Crompton et al., Cancer Research 2015; 75(2):296-305).

This study was performed to investigate whether AKT inhibition during ex vivo TIL expansion could increase the proportion of less differentiated, more stem-like cells with improved cytokine output and functionality.

method

Patient tumors from different indications were received, fragmented and subjected to an expansion protocol for TIL production. During ex vivo expansion, different doses (0.3 μM and 1 μM) of the pan-AKT inhibitor (AKTi) pataxerti were added to the cultures. See Figure 35 for a schematic diagram of the study. The final TIL products were used to assess the expansion potential and phenotypic and functional characteristics of TILs.

result

Figure 36 shows that AKTi treatment maintains TIL expansion and viability without affecting T cell ratios. TILs were left untreated (CTRL, gray bars) or treated with increasing concentrations of pan-AKTi pataseti. Treatments were added only during the REP phase (blue bars) or during pre-REP and REP (green bars). Figure 36A shows the expansion fold and viability of TILs at the end of the 22-day expansion process, and Figure 36B shows in the left panel, middle panel, and right panel, respectively, in cryopreserved cells after the expansion process The appearance rate of CD8+, CD4+ and CD4+(Foxp3+) cells.

Figure 37 shows that AKT inhibition increases the appearance of CD8+ Temra cells. Specifically, Figure 37 shows the appearance of Tcm (CD45RA-CCR7+), Tem (CD45RA-CCR7-) and Temra (CD45+CCR7-) cells in CD8+ (Figure 37A) and CD4+ (Figure 37B) TILs following treatment rate, where *P<0.05.

Figure 38 shows that AKTi treatment increases IL-7R and CXCR3 expression on CD8+ TILs. Cryopreserved control or AKTi-treated TILs were analyzed by flow cytometry, and Figure 38 shows representative histograms and occurrences of IL-7R+ (Figure 38A) and CXCR3+CD8+ (Figure 38B) TILs, where *P<0.05, **P<0.01.

Figure 39 shows that AKT inhibition increases the appearance of CD69-CD39-CD8+ TILs. The frequency of occurrence of CD69 and CD39 subsets on CD8+ TILs was analyzed and CD69 and CD39 single positive and double positive populations as well as single negative and Distribution of double negative population, where *P<0.05, **P<0.01, ***P<0.001.

Figure 40 shows that CD69-CD39-CD8+ TILs are less differentiated. Expression of inhibitory receptors and transcription factors on CD69-CD39- and CD69+CD39+ CD8+ TILs was assessed. Figure 40A shows the frequency of PD1, LAG3, TIM3 and TIGIT and Tbet, Eomes, Batf and TOX on CD69-CD39-CD8+ and CD69+CD39+CD8+ TIL cells. Figure 40B shows representative histograms and frequency of CD62L expression on CD69-CD39-CD8+ and CD69+CD39+CD8+ TILs, where *P<0.05, **P<0.01, ****P<0.0001.

Figure 41 shows that AKTi-treated TILs maintain higher CD69-CD39- cell appearance, lower TOX expression, and higher cytokine output after stimulation. Marker expression was assessed in control and AKTi-treated TILs following overnight stimulation. Cryopreserved controls and TILs treated with 1 μΜ AKTi in pre-REP and REP were stimulated overnight with anti-CD3/CD28 beads at a bead:cell ratio of 1:5. Figure 41A shows the occurrence rate and transcription factor expression of CD69-CD39- and CD69+CD39+ cells on CD8+ TILs, and Figure 41B shows the expression of cytokines on control and AKTi-treated CD8+ TILs, where *P<0.05, **P<0.01, ***P<0.001.

Figure 42 shows that AKTi-treated TILs exhibit increased cytotoxicity in the allogeneic setting, which persists after repeated stimulation. Cytotoxicity of control and AKTi-treated TILs was assessed. In FIG. 42A , cryopreserved controls and TILs treated with 1 μM AKTi in pre-REP and REP were mixed with KILR® THP-1 cells (Eurofins DiscoverX, Fremont, CA, USA) at a 10:1 effector ratio. : Target cell ratios were co-cultured for 24 hours to measure cytotoxicity in the allogeneic setting. In Figure 42B, control and AKTi-treated TILs were stimulated with anti-CD3/CD28 beads at a 1:1 bead:cell ratio every 5 days. Three days after the third stimulation, cells were washed, beads were removed, and cells were co-cultured with KILR THP-1 cells at a 10:1 effector:target cell ratio for 24 hours.

in conclusion

AKTi treatment with a dose of 1 μM resulted in equivalent expansion and survival of TILs compared to controls, but doubled the less differentiated CD39-CD69- cell population. This effect was present even after restimulation and these cells displayed reduced expression of CD38 and the transcription factors T-bet and Tox, indicating a less differentiated and depleted phenotype. Importantly, AKTi treatment caused an increased appearance of IFNγ+TNFα+ CD8+ T cells, which translated into increased cytotoxicity.

AKTi treatment during ex vivo TIL expansion increases the proportion of less differentiated, more memory-like functional TILs. Thus, transient inhibition of AKT signaling during TIL expansion may represent an approach for improving TIL quality and enhancing TIL persistence and therapeutic efficacy in the clinical setting.

The above examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use embodiments of the compositions, systems, and methods of the invention, and are not intended to limit the scope of the inventors' definition of their invention. category. Modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications mentioned in this specification are indicative of the level of skill in the art of those skilled in the art to which this invention pertains.

All headings and section names are used for clarity and reference purposes only and should not be considered limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining aspects from different headings and sections as desired in accordance with the spirit and scope of the invention described herein.

All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The manner incorporated in this article is general.

Various modifications and changes can be made thereto without departing from the spirit and scope of the application, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are provided as examples only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled.

[ FIG. 1 ] : An exemplary Gen 2 (Method 2A) diagram providing an overview of steps A to F.

[ FIG . 2A- FIG. 2C ] : Method flow diagram of an embodiment of Gen 2 (Method 2A) for TIL fabrication.

[ FIG. 3 ] : A diagram showing an example of an exemplary production method of TIL cryopreserved (approximately 22 days).

[ FIG. 4 ] : A diagram showing an example of Gen 2 (Method 2A, ie, a 22-day method for TIL production).

[ FIG. 5 ] : Comparison table of Steps A to F of an exemplary embodiment of Method 1C and Gen 2 for TIL fabrication (Method 2A).

[ FIG. 6 ] : Detailed comparison of an embodiment of method 1C and an embodiment of Gen 2 (method 2A) for TIL fabrication.

[ FIG. 7 ] : Exemplary Gen 3 type TIL manufacturing method.

[ FIGS. 8A-8G ]: A) Shows the comparison between the 2A method (approximately 22-day process) and an example of the Gen 3 process for TIL production (approximately 14-16 day process). B) Exemplary Gen3 process diagram providing an overview of steps A through F (approximately 14-day to 16-day process). C) Provides a diagram of three exemplary Gen 3 processes outlining Steps A to F (approximately 14-day to 16-day processes) for each of the three process variations. D) An exemplary modified Gen 2-like process that provides an overview of steps A to F (approximately 22 day process). E) Shows a comparison between the 2A method (approximately 22 day process) and an example of the Gen 3 process for TIL production (approximately 14-22 day process). F) Combined Gen3 Chart of Exemplary Methods (a) CD39/CD69 Double Negative, (b) CD39/CD69 Double Knockout, or (i) and (ii) TIL Amplification Methods, which Provides an Overview of Steps A to F ( About 14 days to 22 days method). G) Exemplary embodiments of (a) CD39/CD69 double negative, (b) CD39/CD69 double knockout, or combination of (i) and (ii) TIL expansion methods with pre-selection described herein.

[ FIG. 9 ] : Provides a flow chart of experiments regarding the comparability between Gen 2 (Method 2A) and Gen 3 methods.

[ FIG. 10 ] : Shows a comparison between various Gen 2 (Method 2A) and Gen 3.1 method embodiments.

[ FIG. 11 ] : A table describing various features of embodiments of Gen 2, Gen 2.1 and Gen 3.0 methods.

[ FIG. 12 ] : Outline of culture medium conditions of an example of the Gen 3 method (referred to as Gen 3.1).

[ FIG. 13 ] : A table describing various features of embodiments of Gen 2, Gen 2.1 and Gen 3.0 methods.

[ FIG. 14 ] : A table comparing various features of embodiments of Gen 2 and Gen 3.0 processes.

[ FIG. 15 ] : A table providing the use of the medium in various embodiments of the described amplification method.

[ FIG. 16 ] : Schematic diagram of an exemplary embodiment of the Gen 3 method (16-day method).

[ FIG. 17 ] : Schematic diagram of an exemplary embodiment of a method for expanding T cells from hematopoietic malignancies using the Gen 3 expansion platform.

[ FIG. 18 ] : Structures IA and IB are provided. The cylinder system refers to individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF binding domains derived from, for example, 4-1BBL or an antibody that binds 4-1BB, which fold to form a trivalent protein that is then passed through an IgG1-Fc (comprising CH3 and CH2 domains). ) to another trivalent protein, followed by linking the two trivalent proteins together via disulfide bonds (small elongated ovals), thereby stabilizing the structure and providing the ability to connect the intracellular signaling domains of the six receptors to Agonists of signaling proteins that come together to form signaling complexes. A TNFRSF binding domain represented as a cylinder may be a scFv domain comprising, for example, VH and VL chains connected by a linker which may comprise hydrophilic residues and Gly and Ser sequences providing flexibility and Glu providing solubility with Lys.

[ FIG. 19 ] : Schematic diagram of an exemplary embodiment of the Gen 3 method (16-day method).

[ FIG. 20 ] : Provides a method overview of an exemplary embodiment of the Gen 3.1 method (16-day type method).

[ FIG. 21 ] : Schematic diagram of an exemplary embodiment of the Gen 3.1 test method (16-day to 17-day type method).

[ FIG. 22 ] : Schematic diagram of an exemplary embodiment of the Gen 3 method (16-day method).

[ FIG. 23 ] : Comparison table of exemplary Gen 2 and exemplary Gen 3 methods.

[ FIG. 24 ] : Schematic diagram of an exemplary embodiment of a Gen 3 method (16-day/17-day method) preparation schedule.

[ FIG. 25 ] : Schematic diagram of an exemplary embodiment of the Gen 3 method (14-16 day method).

[ FIG . 26A- FIG. 26B ] : Schematic diagram of an exemplary embodiment of the Gen 3 method (16-day type method).

[ FIG. 27 ] : Schematic diagram of an exemplary embodiment of the Gen 3 method (16-day type method).

[ FIG. 28 ] : Comparison of Gen 2, Gen 2.1 and Gen 3 methods (16-day method).

[ FIG. 29 ] : Comparison of Gen 2, Gen 2.1 and Gen 3 methods (method of 16 days).

[ FIG. 30 ] : Gen 3 Example Components.

[ FIG. 31 ] : Flow chart comparison of Gen 3 embodiments (Gen 3.0, Gen 3.1 Control, Gen 3.1 Test).

[ FIG. 32 ] : Shows the components of an exemplary embodiment of the Gen 3 method (16-17 day type method).

[ Figure 33 ] : Acceptance Criteria Form.

[ FIG. 34 ] : A schematic diagram of the workflow in Example 15.

[ FIG. 35 ] : A schematic diagram of the workflow in Example 17.

[ FIGS . 36A- 36B ] : Evaluation of the effect of AKTi treatment on TIL expansion , survival and T cell distribution.

[ FIGS . 37A- 37B ] : Evaluation of T cell subsets in control and AKTi-treated TILs.

[ FIG . 38A- FIG. 38B ] : Assessment of cytokine and chemokine receptor expression on control and AKTi-treated TILs.

[ FIG. 39 ] : Evaluation of the distribution of CD69 and CD39 single positive and double positive populations and single negative and double negative populations in control and AKTi-treated CD8+ TILs.

[ FIG . 40A- FIG. 40B ] : Evaluation of the expression of inhibitory receptors and transcription factors on CD69-CD39- and CD69+ CD39+CD8+ TILs.

[ FIG . 41A- FIG. 41B ] : Evaluation of marker expression in control and AKTi-treated TILs after overnight stimulation.

[ FIG . 42A- FIG. 42B ] : Evaluation of cytotoxicity of control and AKTi-treated TILs.

         
           <![CDATA[ <110> Iovance Biotherapeutics, Inc.]]>
           <![CDATA[ <120> Method for TIL amplification associated with CD39/CD69 selection and gene knockout in tumor infiltrating lymphocytes (TIL)]]>
           <![CDATA[ <130> 116983-5090-TW]]>
           <![CDATA[ <140> 111110412]]>
           <![CDATA[ <141> 2022-03-21]]>
           <![CDATA[ <150> US 63/163,730]]>
           <![CDATA[ <151> 2021-03-19]]>
           <![CDATA[ <150> US 63/255,657]]>
           <![CDATA[ <151> 2021-10-14]]>
           <![CDATA[ <150> US 63/280,536]]>
           <![CDATA[ <151> 2021-11-17]]>
           <![CDATA[ <160> 247 ]]>
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          Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
          1 5 10 15
          Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr
                      20 25 30
          Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
                  35 40 45
          Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
              50 55 60
          Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr
                  115 120 125
          Pro Leu Ala Pro Val Cys Gly Gly Thr Thr Gly Ser Ser Val Thr Leu
              130 135 140
          Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp
          145 150 155 160
          Asn Ser Gly Ser Leu Ser Ser Ser Gly Val His Thr Phe Pro Ala Val Leu
                          165 170 175
          Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser
                      180 185 190
          Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser
                  195 200 205
          Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Pro Lys Ser Cys Asp
              210 215 220
          Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
          225 230 235 240
          Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
                          245 250 255
          Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
                      260 265 270
          Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
                  275 280 285
          Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
              290 295 300
          Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
          305 310 315 320
          Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
                          325 330 335
          Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
                      340 345 350
          Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
                  355 360 365
          Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
              370 375 380
          Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
          385 390 395 400
          Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
                          405 410 415
          Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
                      420 425 430
          Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
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          Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
                      20 25 30
          Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
                  35 40 45
          Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser
              50 55 60
          Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu
          65 70 75 80
          Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr
                          85 90 95
          Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Ala Asp Thr Ala Pro
                      100 105 110
          Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly
                  115 120 125
          Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn
              130 135 140
          Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn
          145 150 155 160
          Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser
                          165 170 175
          Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr
                      180 185 190
          Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe
                  195 200 205
          Asn Arg Asn Glu Cys
              210
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          Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
          1 5 10 15
          His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
                      20 25 30
          Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro
                  35 40 45
          Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Glu Leu
              50 55 60
          Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
          65 70 75 80
          Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
                          85 90 95
          Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
                      100 105 110
          Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser
                  115 120 125
          Ile Ile Ser Thr Leu Thr
              130
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          Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu
          1 5 10 15
          Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn
                      20 25 30
          Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys
                  35 40 45
          Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro
              50 55 60
          Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg
          65 70 75 80
          Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys
                          85 90 95
          Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr
                      100 105 110
          Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile
                  115 120 125
          Ser Thr Leu Thr
              130
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          Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
          1 5 10 15
          Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
                      20 25 30
          Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
                  35 40 45
          Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
              50 55 60
          Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
          65 70 75 80
          Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
                          85 90 95
          Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
                      100 105 110
          Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
                  115 120 125
          Ile Ser Thr Leu Thr
              130
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          1 5 10 15
          Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu
                      20 25 30
          Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile
                  35 40 45
          Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Ser Ser Ser Ser
              50 55 60
          Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln
          65 70 75 80
          Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg
                          85 90 95
          Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys
                      100 105 110
          His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu
                  115 120 125
          Asn Leu Ala Gln Gly Ser Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp
              130 135 140
          Pro Pro Glu Ile Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu
          145 150 155 160
          Gly Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys
                          165 170 175
          Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser
                      180 185 190
          Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr
                  195 200 205
          Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr
              210 215 220
          Glu Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly
          225 230 235 240
          His Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile
                          245 250 255
          Tyr His Phe Val Val Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly
                      260 265 270
          Tyr Arg Ala Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr
                  275 280 285
          His Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly
              290 295 300
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           <![CDATA[ <220>]]>
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          Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Leu Cys Gly
          1 5 10 15
          Ala Val Phe Val Ser Ala Arg Arg Pro Ser Gly Arg Lys Ser Ser Lys
                      20 25 30
          Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu
                  35 40 45
          Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn Val Asn
              50 55 60
          Leu Glu Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala Leu Phe
          65 70 75 80
          Leu Gly Ile His Gly Gly Lys Met Cys Leu Ser Cys Val Lys Ser Gly
                          85 90 95
          Asp Glu Thr Arg Leu Gln Leu Glu Ala Val Asn Ile Thr Asp Leu Ser
                      100 105 110
          Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser
                  115 120 125
          Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu
              130 135 140
          Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser Leu Thr Asn Met Pro
          145 150 155 160
          Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu Ser
                          165 170 175
          Gly Ser Gly Gly Ala Ser Ser Glu Ser Ser Ala Ser Ser Asp Gly Pro
                      180 185 190
          His Pro Val Ile Thr Glu Ser Arg Ala Ser Ser Glu Ser Ser Ala Ser
                  195 200 205
          Ser Asp Gly Pro His Pro Val Ile Thr Glu Ser Arg Glu Pro Lys Ser
              210 215 220
          Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
          225 230 235 240
          Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
                          245 250 255
          Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
                      260 265 270
          His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
                  275 280 285
          Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
              290 295 300
          Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
          305 310 315 320
          Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
                          325 330 335
          Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
                      340 345 350
          Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
                  355 360 365
          Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
              370 375 380
          Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
          385 390 395 400
          Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
                          405 410 415
          Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
                      420 425 430
          Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
                  435 440 445
          Ser Pro Gly Lys
              450
           <![CDATA[ <210> 8]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Mucin domain polypeptide]]>
           <![CDATA[ <400> 8]]>
          Ser Glu Ser Ser Ala Ser Ser Asp Gly Pro His Pro Val Ile Thr Pro
          1 5 10 15
           <![CDATA[ <210> 9]]>
           <![CDATA[ <211> 130]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of recombinant human IL-4 protein]]>
           <![CDATA[ <400> 9]]>
          Met His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn
          1 5 10 15
          Ser Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp
                      20 25 30
          Ile Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg
                  35 40 45
          Ala Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr
              50 55 60
          Arg Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu
          65 70 75 80
          Ile Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly
                          85 90 95
          Leu Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn
                      100 105 110
          Phe Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys
                  115 120 125
          Ser Ser
              130
           <![CDATA[ <210> 10]]>
           <![CDATA[ <211> 153]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of recombinant human IL-7 protein]]>
           <![CDATA[ <400> 10]]>
          Met Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val
          1 5 10 15
          Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly
                      20 25 30
          Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys
                  35 40 45
          Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu
              50 55 60
          Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu
          65 70 75 80
          Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln
                          85 90 95
          Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys
                      100 105 110
          Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp
                  115 120 125
          Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn
              130 135 140
          Lys Ile Leu Met Gly Thr Lys Glu His
          145 150
           <![CDATA[ <210> 11]]>
           <![CDATA[ <211> 115]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of recombinant human IL-15 protein]]>
           <![CDATA[ <400> 11]]>
          Met Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu
          1 5 10 15
          Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
                      20 25 30
          His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu
                  35 40 45
          Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val
              50 55 60
          Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn
          65 70 75 80
          Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn
                          85 90 95
          Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile
                      100 105 110
          Asn Thr Ser
                  115
           <![CDATA[ <210> 12]]>
           <![CDATA[ <211> 132]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of recombinant human IL-21 protein]]>
           <![CDATA[ <400> 12]]>
          Met Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val
          1 5 10 15
          Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro
                      20 25 30
          Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys
                  35 40 45
          Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg
              50 55 60
          Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr
          65 70 75 80
          Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp
                          85 90 95
          Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser
                      100 105 110
          Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly
                  115 120 125
          Ser Glu Asp Ser
              130
           <![CDATA[ <210> 13]]>
           <![CDATA[ <211> 153]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> IL-2 sequence]]>
           <![CDATA[ <400> 13]]>
          Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
          1 5 10 15
          Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Ser Thr Lys Lys Thr Gln Leu
                      20 25 30
          Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile
                  35 40 45
          Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe
              50 55 60
          Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu
          65 70 75 80
          Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys
                          85 90 95
          Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile
                      100 105 110
          Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala
                  115 120 125
          Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe
              130 135 140
          Cys Gln Ser Ile Ile Ser Thr Leu Thr
          145 150
           <![CDATA[ <210> 14]]>
           <![CDATA[ <211> 133]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> IL-2 mutein sequence]]>
           <![CDATA[ <400> 14]]>
          Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
          1 5 10 15
          Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
                      20 25 30
          Asn Pro Lys Leu Thr Ala Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
                  35 40 45
          Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
              50 55 60
          Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
          65 70 75 80
          Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
                          85 90 95
          Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
                      100 105 110
          Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
                  115 120 125
          Ile Ser Thr Leu Thr
              130
           <![CDATA[ <210> 15]]>
           <![CDATA[ <211> 133]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> IL-2 mutein sequence]]>
           <![CDATA[ <400> 15]]>
          Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
          1 5 10 15
          Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
                      20 25 30
          Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys
                  35 40 45
          Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
              50 55 60
          Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
          65 70 75 80
          Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
                          85 90 95
          Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
                      100 105 110
          Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
                  115 120 125
          Ile Ser Thr Leu Thr
              130
           <![CDATA[ <210> 16]]>
           <![CDATA[ <211> 145]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR1_IL-2 of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 16]]>
          Gly Phe Ser Leu Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu
          1 5 10 15
          Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile
                      20 25 30
          Asn Asn Tyr Lys Asn Pro Lys Leu Thr Ala Met Leu Thr Phe Lys Phe
                  35 40 45
          Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu
              50 55 60
          Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys
          65 70 75 80
          Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile
                          85 90 95
          Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala
                      100 105 110
          Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe
                  115 120 125
          Cys Gln Ser Ile Ile Ser Thr Leu Thr Ser Thr Ser Gly Met Ser Val
              130 135 140
          Gly
          145
           <![CDATA[ <210> 17]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR2 of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 17]]>
          Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu Lys Ser
          1 5 10 15
           <![CDATA[ <210> 18]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR3 of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 18]]>
          Ser Met Ile Thr Asn Trp Tyr Phe Asp Val
          1 5 10
           <![CDATA[ <210> 19]]>
           <![CDATA[ <211> 141]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR1_IL-2 kabat of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 19]]>
          Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
          1 5 10 15
          Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
                      20 25 30
          Asn Pro Lys Leu Thr Ala Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
                  35 40 45
          Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
              50 55 60
          Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
          65 70 75 80
          Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
                          85 90 95
          Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
                      100 105 110
          Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
                  115 120 125
          Ile Ser Thr Leu Thr Ser Thr Ser Gly Met Ser Val Gly
              130 135 140
           <![CDATA[ <210> 20]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR2 kabat of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 20]]>
          Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu Lys Ser
          1 5 10 15
           <![CDATA[ <210> 21]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR3 kabat of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 21]]>
          Ser Met Ile Thr Asn Trp Tyr Phe Asp Val
          1 5 10
           <![CDATA[ <210> 22]]>
           <![CDATA[ <211> 142]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR1_IL-2 clothia of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 22]]>
          Gly Phe Ser Leu Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu
          1 5 10 15
          Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile
                      20 25 30
          Asn Asn Tyr Lys Asn Pro Lys Leu Thr Ala Met Leu Thr Phe Lys Phe
                  35 40 45
          Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu
              50 55 60
          Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys
          65 70 75 80
          Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile
                          85 90 95
          Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala
                      100 105 110
          Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe
                  115 120 125
          Cys Gln Ser Ile Ile Ser Thr Leu Thr Ser Thr Ser Gly Met
              130 135 140
           <![CDATA[ <210> 23]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR2 clothia of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 23]]>
          Trp Trp Asp Asp Lys
          1 5
           <![CDATA[ <210> 24]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR3 clothia of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 24]]>
          Ser Met Ile Thr Asn Trp Tyr Phe Asp Val
          1 5 10
           <![CDATA[ <210> 25]]>
           <![CDATA[ <211> 143]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR1_IL-2 IMGT of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 25]]>
          Gly Phe Ser Leu Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu
          1 5 10 15
          Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile
                      20 25 30
          Asn Asn Tyr Lys Asn Pro Lys Leu Thr Ala Met Leu Thr Phe Lys Phe
                  35 40 45
          Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu
              50 55 60
          Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys
          65 70 75 80
          Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile
                          85 90 95
          Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala
                      100 105 110
          Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe
                  115 120 125
          Cys Gln Ser Ile Ile Ser Thr Leu Thr Ser Thr Ser Gly Met Ser
              130 135 140
           <![CDATA[ <210> 26]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR2 IMGT of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 26]]>
          Ile Trp Trp Asp Asp Lys Lys
          1 5
           <![CDATA[ <210> 27]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> HCDR3 IMGT of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 27]]>
          Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val
          1 5 10
           <![CDATA[ <210> 28]]>
           <![CDATA[ <211> 253]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> VH chain of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 28]]>
          Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln
          1 5 10 15
          Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ala Pro Thr
                      20 25 30
          Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu
                  35 40 45
          Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys
              50 55 60
          Leu Thr Ala Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr
          65 70 75 80
          Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu
                          85 90 95
          Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg
                      100 105 110
          Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser
                  115 120 125
          Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val
              130 135 140
          Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr
          145 150 155 160
          Leu Thr Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro
                          165 170 175
          Gly Lys Ala Leu Glu Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys
                      180 185 190
          Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr
                  195 200 205
          Ser Lys Asn Gln Val Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp
              210 215 220
          Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe
          225 230 235 240
          Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
                          245 250
           <![CDATA[ <210> 29]]>
           <![CDATA[ <211> 533]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> IgG.IL2R67A.H1 heavy chain]]>
           <![CDATA[ <400> 29]]>
          Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr
          1 5 10 15
          Ala Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu
                      20 25 30
          Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val
                  35 40 45
          Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu
              50 55 60
          Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr
          65 70 75 80
          Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe
                          85 90 95
          Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr
                      100 105 110
          Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys
                  115 120 125
          Ala Leu Glu Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr
              130 135 140
          Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
          145 150 155 160
          Asn Gln Val Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala
                          165 170 175
          Thr Tyr Tyr Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val
                      180 185 190
          Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
                  195 200 205
          Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
              210 215 220
          Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
          225 230 235 240
          Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
                          245 250 255
          Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val
                      260 265 270
          Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
                  275 280 285
          Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
              290 295 300
          Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
          305 310 315 320
          Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
                          325 330 335
          Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val
                      340 345 350
          Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
                  355 360 365
          Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
              370 375 380
          Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
          385 390 395 400
          Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Ala Ala
                          405 410 415
          Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
                      420 425 430
          Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
                  435 440 445
          Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
              450 455 460
          Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
          465 470 475 480
          Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
                          485 490 495
          Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
                      500 505 510
          Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
                  515 520 525
          Leu Ser Pro Gly Lys
              530
           <![CDATA[ <210> 30]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> LCDR1 kabat of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 30]]>
          Lys Ala Gln Leu Ser Val Gly Tyr Met His
          1 5 10
           <![CDATA[ <210> 31]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> LCDR2 kabat of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 31]]>
          Asp Thr Ser Lys Leu Ala Ser
          1 5
           <![CDATA[ <210> 32]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> LCDR3 kabat of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 32]]>
          Phe Gln Gly Ser Gly Tyr Pro Phe Thr
          1 5
           <![CDATA[ <210> 33]]>
           <![CDATA[ <211> 6]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> LCDR1 chothia of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 33]]>
          Gln Leu Ser Val Gly Tyr
          1 5
           <![CDATA[ <210> 34]]>
           <![CDATA[ <211> 3]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> LCDR2 chothia of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 34]]>
          Asp Thr Ser
          1           
           <![CDATA[ <210> 35]]>
           <![CDATA[ <211> 6]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> LCDR3 chothia of IgG.IL2R67A.H1]]>
           <![CDATA[ <400> 35]]>
          Gly Ser Gly Tyr Pro Phe
          1 5
           <![CDATA[ <210> 36]]>
           <![CDATA[ <211> 106]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> VL Chain]]>
           <![CDATA[ <400> 36]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser Val Gly Tyr Met
                      20 25 30
          His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
                  35 40 45
          Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
              50 55 60
          Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp
          65 70 75 80
          Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
                          85 90 95
          Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
                      100 105
           <![CDATA[ <210> 37]]>
           <![CDATA[ <211> 213]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> light chain]]>
           <![CDATA[ <400> 37]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser Val Gly Tyr Met
                      20 25 30
          His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
                  35 40 45
          Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
              50 55 60
          Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp
          65 70 75 80
          Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
                          85 90 95
          Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
                      100 105 110
          Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
                  115 120 125
          Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
              130 135 140
          Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
          145 150 155 160
          Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
                          165 170 175
          Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
                      180 185 190
          Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
                  195 200 205
          Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 38]]>
           <![CDATA[ <211> 583]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> light chain]]>
           <![CDATA[ <400> 38]]>
          Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln
          1 5 10 15
          Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ala Pro Thr
                      20 25 30
          Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu
                  35 40 45
          Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys
              50 55 60
          Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr
          65 70 75 80
          Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu
                          85 90 95
          Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg
                      100 105 110
          Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser
                  115 120 125
          Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val
              130 135 140
          Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr
          145 150 155 160
          Leu Thr Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro
                          165 170 175
          Gly Lys Ala Leu Glu Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys
                      180 185 190
          Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr
                  195 200 205
          Ser Lys Asn Gln Val Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp
              210 215 220
          Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe
          225 230 235 240
          Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
                          245 250 255
          Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
                      260 265 270
          Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
                  275 280 285
          Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
              290 295 300
          Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
          305 310 315 320
          Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
                          325 330 335
          Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
                      340 345 350
          Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
                  355 360 365
          Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
              370 375 380
          Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
          385 390 395 400
          Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
                          405 410 415
          Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
                      420 425 430
          Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
                  435 440 445
          Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
              450 455 460
          Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
          465 470 475 480
          Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
                          485 490 495
          Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
                      500 505 510
          Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
                  515 520 525
          Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
              530 535 540
          Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
          545 550 555 560
          Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
                          565 570 575
          Leu Ser Leu Ser Pro Gly Lys
                      580
           <![CDATA[ <210> 39]]>
           <![CDATA[ <211> 213]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> light chain]]>
           <![CDATA[ <400> 39]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser Val Gly Tyr Met
                      20 25 30
          His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
                  35 40 45
          Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
              50 55 60
          Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp
          65 70 75 80
          Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
                          85 90 95
          Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
                      100 105 110
          Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
                  115 120 125
          Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
              130 135 140
          Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
          145 150 155 160
          Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
                          165 170 175
          Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
                      180 185 190
          Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
                  195 200 205
          Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 40]]>
           <![CDATA[ <211> 255]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of human 4-1BB]]>
           <![CDATA[ <400> 40]]>
          Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu
          1 5 10 15
          Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro
                      20 25 30
          Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys
                  35 40 45
          Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile
              50 55 60
          Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser
          65 70 75 80
          Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly
                          85 90 95
          Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu
                      100 105 110
          Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln
                  115 120 125
          Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys
              130 135 140
          Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro
          145 150 155 160
          Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala
                          165 170 175
          Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu
                      180 185 190
          Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu
                  195 200 205
          Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
              210 215 220
          Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Thr Gln Glu Glu Asp Gly
          225 230 235 240
          Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
                          245 250 255
           <![CDATA[ <210> 41]]>
           <![CDATA[ <211> 256]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of murine 4-1BB]]>
           <![CDATA[ <400> 41]]>
          Met Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val
          1 5 10 15
          Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln
                      20 25 30
          Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro
                  35 40 45
          Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys
              50 55 60
          Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Ser Thr
          65 70 75 80
          His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro
                          85 90 95
          Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr
                      100 105 110
          Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn
                  115 120 125
          Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg
              130 135 140
          Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro
          145 150 155 160
          Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu
                          165 170 175
          Gly Gly Pro Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala
                      180 185 190
          Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe
                  195 200 205
          Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln
              210 215 220
          Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser
          225 230 235 240
          Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu
                          245 250 255
           <![CDATA[ <210> 42]]>
           <![CDATA[ <211> 441]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain of 4-1BB agonist monoclonal antibody utumumab (PF-05082566)]]>
           <![CDATA[ <400> 42]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr
                      20 25 30
          Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
                      100 105 110
          Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
                  115 120 125
          Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
              130 135 140
          Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
          145 150 155 160
          Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
                          165 170 175
          Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
                      180 185 190
          Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
                  195 200 205
          Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro
              210 215 220
          Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro
          225 230 235 240
          Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
                          245 250 255
          Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp
                      260 265 270
          Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
                  275 280 285
          Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val
              290 295 300
          His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
          305 310 315 320
          Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
                          325 330 335
          Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
                      340 345 350
          Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
                  355 360 365
          Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
              370 375 380
          Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe
          385 390 395 400
          Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
                          405 410 415
          Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
                      420 425 430
          Gln Lys Ser Leu Ser Leu Ser Pro Gly
                  435 440
           <![CDATA[ <210> 43]]>
           <![CDATA[ <211> 214]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain of 4-1BB agonist monoclonal antibody utumumab (PF-05082566)]]>
           <![CDATA[ <400> 43]]>
          Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
          1 5 10 15
          Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala
                      20 25 30
          His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
                  35 40 45
          Gln Asp Lys Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
              50 55 60
          Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
          65 70 75 80
          Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Tyr Thr Gly Phe Gly Ser Leu
                          85 90 95
          Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys
                      100 105 110
          Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln
                  115 120 125
          Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly
              130 135 140
          Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly
          145 150 155 160
          Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala
                          165 170 175
          Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser
                      180 185 190
          Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
                  195 200 205
          Ala Pro Thr Glu Cys Ser
              210
           <![CDATA[ <210> 44]]>
           <![CDATA[ <211> 116]]>
           <![CDATA[ <212> ]]>PRT
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> The heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody utumumab (PF-05082566). ]]>
           <![CDATA[ <400> 44]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr
                      20 25 30
          Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
                      100 105 110
          Thr Val Ser Ser
                  115
           <![CDATA[ <210> 45]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> The light chain variable region (VL) of the 4-1BB agonist monoclonal antibody utumumab (PF-05082566). ]]>
           <![CDATA[ <400> 45]]>
          Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
          1 5 10 15
          Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala
                      20 25 30
          His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
                  35 40 45
          Gln Asp Lys Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
              50 55 60
          Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
          65 70 75 80
          Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Tyr Thr Gly Phe Gly Ser Leu
                          85 90 95
          Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
                      100 105
           <![CDATA[ <210> 46]]>
           <![CDATA[ <211> 6]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR1 of 4-1BB agonist monoclonal antibody utumumab (PF-05082566)]]>
           <![CDATA[ <400> 46]]>
          Ser Thr Tyr Trp Ile Ser
          1 5
           <![CDATA[ <210> 47]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR2 of 4-1BB agonist monoclonal antibody utumumab (PF-05082566)]]>
           <![CDATA[ <400> 47]]>
          Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 48]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR3 of 4-1BB agonist monoclonal antibody utumumab (PF-05082566)]]>
           <![CDATA[ <400> 48]]>
          Arg Gly Tyr Gly Ile Phe Asp Tyr
          1 5
           <![CDATA[ <210> 49]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> 4-1BB agonist]]> light chain CDR1 of monoclonal antibody utumumab (PF-05082566)
           <![CDATA[ <400> 49]]>
          Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala His
          1 5 10
           <![CDATA[ <210> 50]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 of 4-1BB agonist monoclonal antibody utumumab (PF-05082566)]]>
           <![CDATA[ <400> 50]]>
          Gln Asp Lys Asn Arg Pro Ser
          1 5
           <![CDATA[ <210> 51]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 of 4-1BB agonist monoclonal antibody utumumab (PF-05082566)]]>
           <![CDATA[ <400> 51]]>
          Ala Thr Tyr Thr Gly Phe Gly Ser Leu Ala Val
          1 5 10
           <![CDATA[ <210> 52]]>
           <![CDATA[ <211> 448]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain of 4-1BB agonist monoclonal antibody Urelumab (BMS-663513)]]>
           <![CDATA[ <400> 52]]>
          Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu
          1 5 10 15
          Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr
                      20 25 30
          Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile
                  35 40 45
          Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu
              50 55 60
          Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
          65 70 75 80
          Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
                          85 90 95
          Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly
                      100 105 110
          Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
                  115 120 125
          Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
              130 135 140
          Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
          145 150 155 160
          Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
                          165 170 175
          Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
                      180 185 190
          Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
                  195 200 205
          Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
              210 215 220
          Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
          225 230 235 240
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                          245 250 255
          Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
                      260 265 270
          Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
                  275 280 285
          Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
              290 295 300
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
          305 310 315 320
          Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
                          325 330 335
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                      340 345 350
          Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
                  355 360 365
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
              370 375 380
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
          385 390 395 400
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
                          405 410 415
          Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                      420 425 430
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
                  435 440 445
           <![CDATA[ <210> 53]]>
           <![CDATA[ <211> 216]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513)]]>
           <![CDATA[ <400> 53]]>
          Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
                  35 40 45
          Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
          65 70 75 80
          Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
                          85 90 95
          Ala Leu Thr Phe Cys Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val
                      100 105 110
          Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
                  115 120 125
          Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr Pro Arg
              130 135 140
          Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
          145 150 155 160
          Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
                          165 170 175
          Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
                      180 185 190
          Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
                  195 200 205
          Lys Ser Phe Asn Arg Gly Glu Cys
              210 215
           <![CDATA[ <210> 54]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy Chain Variable Region (VH) of 4-1BB Agonist Monoclonal Antibody Urelumab (BMS-663513)]]>
           <![CDATA[ <400> 54]]>
          Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp
          1 5 10 15
          Val Leu Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys
                      20 25 30
          Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe
                  35 40 45
          Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu
              50 55 60
          Glu Trp Ile Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro
          65 70 75 80
          Ser Leu Glu Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
                          85 90 95
          Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
                      100 105 110
          Tyr Cys Ala Arg Asp Tyr Gly Pro
                  115 120
           <![CDATA[ <210> 55]]>
           <![CDATA[ <211> 110]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513)]]>
           <![CDATA[ <400> 55]]>
          Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Leu Trp Leu Pro
          1 5 10 15
          Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser
                      20 25 30
          Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
                  35 40 45
          Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
              50 55 60
          Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
          65 70 75 80
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
                          85 90 95
          Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
                      100 105 110
           <![CDATA[ <210> 56]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR1 of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513)]]>
           <![CDATA[ <400> 56]]>
          Gly Tyr Tyr Trp Ser
          1 5
           <![CDATA[ <210> 57]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR2 of the 4-1BB agonist monoclonal antibody Urelumab (BMS-663513)]]>
           <![CDATA[ <400> 57]]>
          Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser
          1 5 10 15
           <![CDATA[ <210> 58]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR3 of the 4-1BB agonist monoclonal antibody Urelumab (BMS-663513)]]>
           <![CDATA[ <400> 58]]>
          Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu
          1 5 10
           <![CDATA[ <210> 59]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213]]>> Artificial Sequence]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Light chain CDR1 of 4-1BB agonist monoclonal antibody Urelumab (BMS-663513)]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;400&gt;59]]&gt;
           <br/>
           <br/> <![CDATA[Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala
          1 5 10
           <![CDATA[ <210> 60]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513)]]>
           <![CDATA[ <400> 60]]>
          Asp Ala Ser Asn Arg Ala Thr
          1 5
           <![CDATA[ <210> 61]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 of the 4-1BB agonist monoclonal antibody usrelumab (BMS-663513)]]>
           <![CDATA[ <400> 61]]>
          Gln Gln Arg Ser Asp Trp Pro Pro Ala Leu Thr
          1 5 10
           <![CDATA[ <210> 62]]>
           <![CDATA[ <211> 230]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Fc domain of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 62]]>
          Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
          1 5 10 15
          Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
                      20 25 30
          Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
                  35 40 45
          Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
              50 55 60
          Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
          65 70 75 80
          Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
                          85 90 95
          Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
                      100 105 110
          Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
                  115 120 125
          Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
              130 135 140
          Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
          145 150 155 160
          Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
                          165 170 175
          Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
                      180 185 190
          Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
                  195 200 205
          Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
              210 215 220
          Ser Leu Ser Pro Gly Lys
          225 230
           <![CDATA[ <210> 63]]>
           <![CDATA[ <211> 22]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 63]]>
          Gly Gly Pro Gly Ser Ser Lys Ser Cys Asp Lys Thr His Thr Cys Pro
          1 5 10 15
          Pro Cys Pro Ala Pro Glu
                      20
           <![CDATA[ <210> 64]]>
           <![CDATA[ <211> 22]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 64]]>
          Gly Gly Ser Gly Ser Ser Ser Lys Ser Cys Asp Lys Thr His Thr Cys Pro
          1 5 10 15
          Pro Cys Pro Ala Pro Glu
                      20
           <![CDATA[ <210> 65]]>
           <![CDATA[ <211> 27]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 65]]>
          Gly Gly Pro Gly Ser Ser Ser Ser Ser Ser Ser Ser Lys Ser Cys Asp Lys
          1 5 10 15
          Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
                      20 25
           <![CDATA[ <210> 66]]>
           <![CDATA[ <211> 27]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 66]]>
          Gly Gly Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Lys Ser Cys Asp Lys
          1 5 10 15
          Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
                      20 25
           <![CDATA[ <210> 67]]>
           <![CDATA[ <211> 29]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 67]]>
          Gly Gly Pro Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Lys Ser Cys
          1 5 10 15
          Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
                      20 25
           <![CDATA[ <210> 68]]>
           <![CDATA[ <211> 29]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 68]]>
          Gly Gly Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Lys Ser Cys
          1 5 10 15
          Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
                      20 25
           <![CDATA[ <210> 69]]>
           <![CDATA[ <211> ]]> 23
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 69]]>
          Gly Gly Pro Gly Ser Ser Gly Ser Gly Ser Asp Lys Thr His Thr Cys
          1 5 10 15
          Pro Pro Cys Pro Ala Pro Glu
                      20
           <![CDATA[ <210> 70]]>
           <![CDATA[ <211> 23]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 70]]>
          Gly Gly Pro Gly Ser Ser Gly Ser Gly Ser Asp Lys Thr His Thr Cys
          1 5 10 15
          Pro Pro Cys Pro Ala Pro Glu
                      20
           <![CDATA[ <210> 71]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 71]]>
          Gly Gly Pro Ser Ser Ser Gly Ser Asp Lys Thr His Thr Cys Pro Pro
          1 5 10 15
          Cys Pro Ala Pro Glu
                      20
           <![CDATA[ <210> 72]]>
           <![CDATA[ <211> 25]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 72]]>
          Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Gly Ser Asp Lys Thr His
          1 5 10 15
          Thr Cys Pro Pro Cys Pro Ala Pro Glu
                      20 25
           <![CDATA[ <210> 73]]>
           <![CDATA[ <211> 246]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223]]>> Fc domain of TNFRSF agonist fusion protein]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;400&gt;73]]&gt;
           <br/>
           <br/> <![CDATA[Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
          1 5 10 15
          Ala Gly Asn Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
                      20 25 30
          Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
                  35 40 45
          Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
              50 55 60
          Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
          65 70 75 80
          Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
                          85 90 95
          Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
                      100 105 110
          Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
                  115 120 125
          Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
              130 135 140
          Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
          145 150 155 160
          Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
                          165 170 175
          Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
                      180 185 190
          Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
                  195 200 205
          Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
              210 215 220
          Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
          225 230 235 240
          Leu Ser Leu Ser Pro Gly
                          245
           <![CDATA[ <210> 74]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 74]]>
          Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
          1 5 10
           <![CDATA[ <210> 75]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 75]]>
          Ser Ser Ser Ser Ser Ser Ser Gly Ser Gly Ser Gly Ser
          1 5 10
           <![CDATA[ <210> 76]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker of TNFRSF agonist fusion protein]]>
           <![CDATA[ <400> 76]]>
          Ser Ser Ser Ser Ser Ser Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
          1 5 10 15
           <![CDATA[ <210> 77]]>
           <![CDATA[ <211> 254]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> 4-1BB ligand (4-1BBL) amino acid sequence]]>
           <![CDATA[ <400> 77]]>
          Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro
          1 5 10 15
          Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val
                      20 25 30
          Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe
                  35 40 45
          Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser
              50 55 60
          Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp
          65 70 75 80
          Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val
                          85 90 95
          Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp
                      100 105 110
          Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu
                  115 120 125
          Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe
              130 135 140
          Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser
          145 150 155 160
          Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala
                          165 170 175
          Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala
                      180 185 190
          Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala
                  195 200 205
          Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His
              210 215 220
          Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val
          225 230 235 240
          Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu
                          245 250
           <![CDATA[ <210> 78]]>
           <![CDATA[ <211> 168]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Soluble fraction of 4-1BBL polypeptide]]>
           <![CDATA[ <400> 78]]>
          Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu
          1 5 10 15
          Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val
                      20 25 30
          Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val
                  35 40 45
          Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg
              50 55 60
          Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His
          65 70 75 80
          Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr
                          85 90 95
          Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly
                      100 105 110
          Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val
                  115 120 125
          His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln
              130 135 140
          Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala
          145 150 155 160
          Gly Leu Pro Ser Pro Arg Ser Glu
                          165
           <![CDATA[ <210> 79]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of 4-1BB agonist antibody 4B4-1-1 version 1]]>
           <![CDATA[ <400> 79]]>
          Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Ser Tyr
                      20 25 30
          Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Val Leu Glu Trp Ile
                  35 40 45
          Gly Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe
              50 55 60
          Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Ser Phe Thr Thr Ala Arg Gly Phe Ala Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser
                  115
           <![CDATA[ <210> 80]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of 4-1BB agonist antibody 4B4-1-1 version 1]]>
           <![CDATA[ <400> 80]]>
          Asp Ile Val Met Thr Gln Ser Pro Ala Thr Gln Ser Val Thr Pro Gly
          1 5 10 15
          Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr
                      20 25 30
          Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
                  35 40 45
          Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro
          65 70 75 80
          Glu Asp Val Gly Val Tyr Tyr Cys Gln Asp Gly His Ser Phe Pro Pro
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
                      100 105
           <![CDATA[ <210> 81]]>
           <![CDATA[ <211> 119]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of 4-1BB agonist antibody 4B4-1-1 version 2]]>
           <![CDATA[ <400> 81]]>
          Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Ser Tyr
                      20 25 30
          Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Val Leu Glu Trp Ile
                  35 40 45
          Gly Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe
              50 55 60
          Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Ser Phe Thr Thr Ala Arg Gly Phe Ala Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser Ala
                  115
           <![CDATA[ <210> 82]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <21]]>3> Artificial sequence]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Light chain variable region (VL) of 4-1BB agonist antibody 4B4-1-1 version 2]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;400&gt;82]]&gt;
           <br/>
           <br/> <![CDATA[Asp Ile Val Met Thr Gln Ser Pro Ala Thr Gln Ser Val Thr Pro Gly
          1 5 10 15
          Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr
                      20 25 30
          Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
                  35 40 45
          Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro
          65 70 75 80
          Glu Asp Val Gly Val Tyr Tyr Cys Gln Asp Gly His Ser Phe Pro Pro
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 83]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of 4-1BB agonist antibody H39E3-2]]>
           <![CDATA[ <400> 83]]>
          Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
          1 5 10 15
          Ala His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
                      20 25 30
          Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
                  35 40 45
          Ser Asp Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
              50 55 60
          Glu Trp Val Ala Asp Ile Lys Asn Asp Gly Ser Tyr Thr Asn Tyr Ala
          65 70 75 80
          Pro Ser Leu Thr Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
                          85 90 95
          Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
                      100 105 110
          Tyr Tyr Cys Ala Arg Glu Leu Thr
                  115 120
           <![CDATA[ <210> 84]]>
           <![CDATA[ <211> 109]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of 4-1BB agonist antibody H39E3-2]]>
           <![CDATA[ <400> 84]]>
          Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Leu Trp Leu Pro
          1 5 10 15
          Asp Thr Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala
                      20 25 30
          Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser
                  35 40 45
          Leu Leu Ser Ser Gly Asn Gln Lys Asn Tyr Leu Trp Tyr Gln Gln Lys
              50 55 60
          Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Arg Gln
          65 70 75 80
          Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
                          85 90 95
          Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
                      100 105
           <![CDATA[ <210> 85]]>
           <![CDATA[ <211> 277]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of human OX40]]>
           <![CDATA[ <400> 85]]>
          Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu
          1 5 10 15
          Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val
                      20 25 30
          Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro
                  35 40 45
          Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys
              50 55 60
          Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Ser Lys Pro
          65 70 75 80
          Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys
                          85 90 95
          Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly
                      100 105 110
          Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys
                  115 120 125
          Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp
              130 135 140
          Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn
          145 150 155 160
          Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro
                          165 170 175
          Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr
                      180 185 190
          Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu
                  195 200 205
          Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val
              210 215 220
          Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu
          225 230 235 240
          Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly
                          245 250 255
          Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser
                      260 265 270
          Thr Leu Ala Lys Ile
                  275
           <![CDATA[ <210> 86]]>
           <![CDATA[ <211> 272]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of murine OX40]]>
           <![CDATA[ <400> 86]]>
          Met Tyr Val Trp Val Gln Gln Pro Thr Ala Leu Leu Leu Leu Gly Leu
          1 5 10 15
          Thr Leu Gly Val Thr Ala Arg Arg Leu Asn Cys Val Lys His Thr Tyr
                      20 25 30
          Pro Ser Gly His Lys Cys Cys Arg Glu Cys Gln Pro Gly His Gly Met
                  35 40 45
          Val Ser Arg Cys Asp His Thr Arg Asp Thr Leu Cys His Pro Cys Glu
              50 55 60
          Thr Gly Phe Tyr Asn Glu Ala Val Asn Tyr Asp Thr Cys Lys Gln Cys
          65 70 75 80
          Thr Gln Cys Asn His Arg Ser Gly Ser Glu Leu Lys Gln Asn Cys Thr
                          85 90 95
          Pro Thr Gln Asp Thr Val Cys Arg Cys Arg Pro Gly Thr Gln Pro Arg
                      100 105 110
          Gln Asp Ser Gly Tyr Lys Leu Gly Val Asp Cys Val Pro Cys Pro Pro
                  115 120 125
          Gly His Phe Ser Pro Gly Asn Asn Gln Ala Cys Lys Pro Trp Thr Asn
              130 135 140
          Cys Thr Leu Ser Gly Lys Gln Thr Arg His Pro Ala Ser Asp Ser Leu
          145 150 155 160
          Asp Ala Val Cys Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp Glu
                          165 170 175
          Thr Gln Arg Pro Thr Phe Arg Pro Thr Thr Val Gln Ser Thr Thr Val
                      180 185 190
          Trp Pro Arg Thr Ser Glu Leu Pro Ser Pro Pro Thr Leu Val Thr Pro
                  195 200 205
          Glu Gly Pro Ala Phe Ala Val Leu Leu Gly Leu Gly Leu Gly Leu Leu
              210 215 220
          Ala Pro Leu Thr Val Leu Leu Ala Leu Tyr Leu Leu Arg Lys Ala Trp
          225 230 235 240
          Arg Leu Pro Asn Thr Pro Lys Pro Cys Trp Gly Asn Ser Phe Arg Thr
                          245 250 255
          Pro Ile Gln Glu Glu His Thr Asp Ala His Phe Thr Leu Ala Lys Ile
                      260 265 270
           <![CDATA[ <210> 87]]>
           <![CDATA[ <211> 451]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain of OX40 agonist monoclonal antibody tavoximab (MEDI-0562)]]>
           <![CDATA[ <400> 87]]>
          Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
          1 5 10 15
          Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser Ser Gly
                      20 25 30
          Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Ile
                  35 40 45
          Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys
              50 55 60
          Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu
          65 70 75 80
          Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
                          85 90 95
          Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
                  115 120 125
          Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
              130 135 140
          Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
          145 150 155 160
          Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
                          165 170 175
          Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
                      180 185 190
          Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
                  195 200 205
          Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
              210 215 220
          Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
          225 230 235 240
          Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
                          245 250 255
          Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
                      260 265 270
          Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
                  275 280 285
          His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
              290 295 300
          Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
          305 310 315 320
          Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
                          325 330 335
          Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
                      340 345 350
          Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
                  355 360 365
          Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
              370 375 380
          Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
          385 390 395 400
          Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
                          405 410 415
          Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
                      420 425 430
          His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
                  435 440 445
          Pro Gly Lys
              450
           <![CDATA[ <210> 88]]>
           <![CDATA[ <211> 214]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562)]]>
           <![CDATA[ <400> 88]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
                      20 25 30
          Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
                      100 105 110
          Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
                  115 120 125
          Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
              130 135 140
          Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
          145 150 155 160
          Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
                          165 170 175
          Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
                      180 185 190
          Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
                  195 200 205
          Phe Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 89]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562)]]>
           <![CDATA[ <400> 89]]>
          Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
          1 5 10 15
          Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser Ser Gly
                      20 25 30
          Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Ile
                  35 40 45
          Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys
              50 55 60
          Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu
          65 70 75 80
          Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
                          85 90 95
          Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr
                  115
           <![CDATA[ <210> 90]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of the OX40 agonist monoclonal antibody tavoximab (MEDI-0562)]]>
           <![CDATA[ <400> 90]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
                      20 25 30
          Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 91]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR1 of OX40 agonist monoclonal antibody tavoximab (MEDI-0562)]]>
           <![CDATA[ <400> 91]]>
          Gly Ser Phe Ser Ser Gly Tyr Trp Asn
          1 5
           <![CDATA[ <210> 92]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR2 of OX40 agonist monoclonal antibody tavoximab (MEDI-0562)]]>
           <![CDATA[ <400> 92]]>
          Tyr Ile Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His
          1 5 10
           <![CDATA[ <210> 93]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR3 of OX40 agonist monoclonal antibody tavoximab (MEDI-0562)]]>
           <![CDATA[ <400> 93]]>
          Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr
          1 5 10
           <![CDATA[ <210> 94]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 of OX40 agonist monoclonal antibody tavoximab (MEDI-0562)]]>
           <![CDATA[ <400> 94]]>
          Gln Asp Ile Ser Asn Tyr Leu Asn
          1 5
           <![CDATA[ <210> 95]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 of OX40 agonist monoclonal antibody tavoximab (MEDI-0562)]]>
           <![CDATA[ <400> 95]]>
          Leu Leu Ile Tyr Tyr Thr Ser Lys Leu His Ser
          1 5 10
           <![CDATA[ <210> 96]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 of OX40 agonist monoclonal antibody tavoximab (MEDI-0562)]]>
           <![CDATA[ <400> 96]]>
          Gln Gln Gly Ser Ala Leu Pro Trp
          1 5
           <![CDATA[ <210> 97]]>
           <![CDATA[ <211> 444]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain of OX40 agonist monoclonal antibody 11D4]]>
           <![CDATA[ <400> 97]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
                      20 25 30
          Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Glu Ser Gly Trp Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr
                      100 105 110
          Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
                  115 120 125
          Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
              130 135 140
          Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
          145 150 155 160
          Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
                          165 170 175
          Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser
                      180 185 190
          Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
                  195 200 205
          Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys
              210 215 220
          Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe
          225 230 235 240
          Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
                          245 250 255
          Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe
                      260 265 270
          Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
                  275 280 285
          Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr
              290 295 300
          Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
          305 310 315 320
          Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
                          325 330 335
          Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
                      340 345 350
          Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
                  355 360 365
          Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
              370 375 380
          Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser
          385 390 395 400
          Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
                          405 410 415
          Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
                      420 425 430
          Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                  435 440
           <![CDATA[ <210> 98]]>
           <![CDATA[ <211> 180]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain of OX40 agonist monoclonal antibody 11D4]]>
           <![CDATA[ <400> 98]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ser Trp
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
                  35 40 45
          Tyr Ala Ala Ser Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
                      100 105 110
          Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
                  115 120 125
          Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
              130 135 140
          Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
          145 150 155 160
          Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
                          165 170 175
          Ser Thr Leu Thr
                      180
           <![CDATA[ <210> 99]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of OX40 agonist monoclonal antibody 11D4]]>
           <![CDATA[ <400> 99]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
                      20 25 30
          Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Glu Ser Gly Trp Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr
                      100 105 110
          Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 100]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of OX40 agonist monoclonal antibody 11D4]]>
           <![CDATA[ <400> 100]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ser Trp
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
                  35 40 45
          Tyr Ala Ala Ser Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
                      100 105
           <![CDATA[ <210> 101]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain of OX40 agonist monoclonal antibody 11D4]]>CDR1
           <![CDATA[ <400> 101]]>
          Ser Tyr Ser Met Asn
          1 5
           <![CDATA[ <210> 102]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR2 of OX40 agonist monoclonal antibody 11D4]]>
           <![CDATA[ <400> 102]]>
          Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 103]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR3 of OX40 agonist monoclonal antibody 11D4]]>
           <![CDATA[ <400> 103]]>
          Glu Ser Gly Trp Tyr Leu Phe Asp Tyr
          1 5
           <![CDATA[ <210> 104]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 of OX40 agonist monoclonal antibody 11D4]]>
           <![CDATA[ <400> 104]]>
          Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala
          1 5 10
           <![CDATA[ <210> 105]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 of OX40 agonist monoclonal antibody 11D4]]>
           <![CDATA[ <400> 105]]>
          Ala Ala Ser Ser Leu Gln Ser
          1 5
           <![CDATA[ <210> 106]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 of OX40 agonist monoclonal antibody 11D4]]>
           <![CDATA[ <400> 106]]>
          Gln Gln Tyr Asn Ser Tyr Pro Pro Thr
          1 5
           <![CDATA[ <210> 107]]>
           <![CDATA[ <211> 450]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain of OX40 agonist monoclonal antibody 18D8]]>
           <![CDATA[ <400> 107]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr
                      20 25 30
          Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
                          85 90 95
          Ala Lys Asp Gln Ser Thr Ala Asp Tyr Tyr Phe Tyr Tyr Gly Met Asp
                      100 105 110
          Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
                  115 120 125
          Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
              130 135 140
          Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
          145 150 155 160
          Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
                          165 170 175
          Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val
                      180 185 190
          Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn
                  195 200 205
          Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg
              210 215 220
          Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly
          225 230 235 240
          Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
                          245 250 255
          Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
                      260 265 270
          Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
                  275 280 285
          Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg
              290 295 300
          Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys
          305 310 315 320
          Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu
                          325 330 335
          Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
                      340 345 350
          Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
                  355 360 365
          Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
              370 375 380
          Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met
          385 390 395 400
          Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
                          405 410 415
          Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
                      420 425 430
          Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                  435 440 445
          Gly Lys
              450
           <![CDATA[ <210> 108]]>
           <![CDATA[ <211> 213]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain of OX40 agonist monoclonal antibody 18D8]]>
           <![CDATA[ <400> 108]]>
          Glu Ile Val Val Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
                  35 40 45
          Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
          65 70 75 80
          Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr
                          85 90 95
          Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
                      100 105 110
          Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
                  115 120 125
          Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
              130 135 140
          Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
          145 150 155 160
          Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
                          165 170 175
          Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
                      180 185 190
          Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
                  195 200 205
          Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 109]]>
           <![CDATA[ <211> 124]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy Chain Variable Region (VH) of OX40 Agonist Monoclonal Antibody 18D8]]>
           <![CDATA[ <400> 109]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr
                      20 25 30
          Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
                          85 90 95
          Ala Lys Asp Gln Ser Thr Ala Asp Tyr Tyr Phe Tyr Tyr Gly Met Asp
                      100 105 110
          Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 110]]>
           <![CDATA[ <211> 106]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of OX40 agonist monoclonal antibody 18D8]]>
           <![CDATA[ <400> 110]]>
          Glu Ile Val Val Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
                  35 40 45
          Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
          65 70 75 80
          Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr
                          85 90 95
          Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
                      100 105
           <![CDATA[ <210> 111]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR1 of OX40 agonist monoclonal antibody 18D8]]>
           <![CDATA[ <400> 111]]>
          Asp Tyr Ala Met His
          1 5
           <![CDATA[ <210> 112]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR2 of OX40 agonist monoclonal antibody 18D8]]>
           <![CDATA[ <400> 112]]>
          Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 113]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR3 of OX40 agonist monoclonal antibody 18D8]]>
           <![CDATA[ <400> 113]]>
          Asp Gln Ser Thr Ala Asp Tyr Tyr Phe Tyr Tyr Gly Met Asp Val
          1 5 10 15
           <![CDATA[ <210> 114]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 of OX40 agonist monoclonal antibody 18D8]]>
           <![CDATA[ <400> 114]]>
          Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala
          1 5 10
           <![CDATA[ <210> 115]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 of OX40 agonist monoclonal antibody 18D8]]>
           <![CDATA[ <400> 115]]>
          Asp Ala Ser Asn Arg Ala Thr
          1 5
           <![CDATA[ <210> 116]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 of OX40 agonist monoclonal antibody 18D8]]>
           <![CDATA[ <400> 116]]>
          Gln Gln Arg Ser Asn Trp Pro Thr
          1 5
           <![CDATA[ <210> 117]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of OX40 agonist monoclonal antibody Hu119-122]]>
           <![CDATA[ <400> 117]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Tyr Glu Phe Pro Ser His
                      20 25 30
          Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val
                  35 40 45
          Ala Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met
              50 55 60
          Glu Arg Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Met Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 118]]>
           <![CDATA[ <211> 111]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of OX40 agonist monoclonal antibody Hu119-122]]>
           <![CDATA[ <400> 118]]>
          Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser
                      20 25 30
          Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
                  35 40 45
          Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
              50 55 60
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65 70 75 80
          Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg
                          85 90 95
          Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
                      100 105 110
           <![CDATA[ <210> 119]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR1 of OX40 agonist monoclonal antibody Hu119-122]]>
           <![CDATA[ <400> 119]]>
          Ser His Asp Met Ser
          1 5
           <![CDATA[ <210> 120]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR2 of OX40 agonist monoclonal antibody Hu119-122]]>
           <![CDATA[ <400> 120]]>
          Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met Glu
          1 5 10 15
          Arg
           <![CDATA[ <210> 121]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR3 of OX40 agonist monoclonal antibody Hu119-122]]>
           <![CDATA[ <400> 121]]>
          His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr
          1 5 10
           <![CDATA[ <210> 122]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 of OX40 agonist monoclonal antibody Hu119-122]]>
           <![CDATA[ <400> 122]]>
          Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His
          1 5 10 15
           <![CDATA[ <210> 123]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 of OX40 agonist monoclonal antibody Hu119-122]]>
           <![CDATA[ <400> 123]]>
          Leu Ala Ser Asn Leu Glu Ser
          1 5
           <![CDATA[ <210> 124]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 of OX40 agonist monoclonal antibody Hu119-122]]>
           <![CDATA[ <400> 124]]>
          Gln His Ser Arg Glu Leu Pro Leu Thr
          1 5
           <![CDATA[ <210> 125]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of OX40 agonist monoclonal antibody Hu106-222]]>
           <![CDATA[ <400> 125]]>
          Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
                      20 25 30
          Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
                  35 40 45
          Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
              50 55 60
          Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp
                      100 105 110
          Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 126]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212]]>> PRT]]&gt;
           <br/> &lt;![CDATA[ &lt;213&gt; Artificial Sequence]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;220&gt;]]&gt;
           <br/> &lt;![CDATA[ &lt;223&gt; Light chain variable region (VL) of OX40 agonist monoclonal antibody Hu106-222]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;400&gt;126]]&gt;
           <br/>
           <br/> <![CDATA[Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
                      20 25 30
          Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
                      100 105
           <![CDATA[ <210> 127]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR1 of OX40 agonist monoclonal antibody Hu106-222]]>
           <![CDATA[ <400> 127]]>
          Asp Tyr Ser Met His
          1 5
           <![CDATA[ <210> 128]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR2 of OX40 agonist monoclonal antibody Hu106-222]]>
           <![CDATA[ <400> 128]]>
          Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 129]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain CDR3 of OX40 agonist monoclonal antibody Hu106-222]]>
           <![CDATA[ <400> 129]]>
          Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr
          1 5 10
           <![CDATA[ <210> 130]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 of OX40 agonist monoclonal antibody Hu106-222]]>
           <![CDATA[ <400> 130]]>
          Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala
          1 5 10
           <![CDATA[ <210> 131]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223]]>> Light chain CDR3 of OX40 agonist monoclonal antibody Hu106-222]]&gt;
           <br/>
           <br/> &lt;![CDATA[ &lt;400&gt;131]]&gt;
           <br/>
           <br/> <![CDATA[Ser Ala Ser Tyr Leu Tyr Thr
          1 5
           <![CDATA[ <210> 132]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 of OX40 agonist monoclonal antibody Hu106-222]]>
           <![CDATA[ <400> 132]]>
          Gln Gln His Tyr Ser Thr Pro Arg Thr
          1 5
           <![CDATA[ <210> 133]]>
           <![CDATA[ <211> 183]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> OX40 ligand (OX40L) amino acid sequence]]>
           <![CDATA[ <400> 133]]>
          Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg
          1 5 10 15
          Pro Arg Phe Glu Arg Asn Lys Leu Leu Leu Val Ala Ser Val Ile Gln
                      20 25 30
          Gly Leu Gly Leu Leu Leu Cys Phe Thr Tyr Ile Cys Leu His Phe Ser
                  35 40 45
          Ala Leu Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val
              50 55 60
          Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln
          65 70 75 80
          Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn
                          85 90 95
          Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu
                      100 105 110
          Val Asn Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln
                  115 120 125
          Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr
              130 135 140
          Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu
          145 150 155 160
          Asp Asp Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn
                          165 170 175
          Pro Gly Glu Phe Cys Val Leu
                      180
           <![CDATA[ <210> 134]]>
           <![CDATA[ <211> 131]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Soluble fraction of OX40L polypeptide]]>
           <![CDATA[ <400> 134]]>
          Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe Thr Glu
          1 5 10 15
          Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu Asp Glu
                      20 25 30
          Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn Cys Asp Gly Phe
                  35 40 45
          Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu Val Asn Ile Ser
              50 55 60
          Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln Leu Lys Lys Val
          65 70 75 80
          Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys Asp Lys
                          85 90 95
          Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp Phe His
                      100 105 110
          Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn Pro Gly Glu Phe
                  115 120 125
          Cys Val Leu
              130
           <![CDATA[ <210> 135]]>
           <![CDATA[ <211> 128]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Alternative soluble portion of OX40L polypeptide]]>
           <![CDATA[ <400> 135]]>
          Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe Thr Glu Tyr Lys Lys
          1 5 10 15
          Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu Asp Glu Ile Met Lys
                      20 25 30
          Val Gln Asn Asn Ser Val Ile Ile Asn Cys Asp Gly Phe Tyr Leu Ile
                  35 40 45
          Ser Leu Lys Gly Tyr Phe Ser Gln Glu Val Asn Ile Ser Leu His Tyr
              50 55 60
          Gln Lys Asp Glu Glu Pro Leu Phe Gln Leu Lys Lys Val Arg Ser Val
          65 70 75 80
          Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys Asp Lys Val Tyr Leu
                          85 90 95
          Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp Phe His Val Asn Gly
                      100 105 110
          Gly Glu Leu Ile Leu Ile His Gln Asn Pro Gly Glu Phe Cys Val Leu
                  115 120 125
           <![CDATA[ <210> 136]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of OX40 agonist monoclonal antibody 008]]>
           <![CDATA[ <400> 136]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Lys Asp Arg Tyr Ser Gln Val His Tyr Ala Leu Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser
                  115 120
           <![CDATA[ <210> 137]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of OX40 agonist monoclonal antibody 008]]>
           <![CDATA[ <400> 137]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Pro Val Thr Pro Gly
          1 5 10 15
          Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
                      20 25 30
          Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ala Gly Gln Ser
                  35 40 45
          Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
              50 55 60
          Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
          65 70 75 80
          Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr
                          85 90 95
          Tyr Asn His Pro Thr Thr Phe Gly Gln Gly Thr Lys
                      100 105
           <![CDATA[ <210> 138]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of OX40 agonist monoclonal antibody 011]]>
           <![CDATA[ <400> 138]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Ser Ile Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Arg Lys Gly
              50 55 60
          Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
          65 70 75 80
          Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
                          85 90 95
          Asp Arg Tyr Phe Arg Gln Gln Asn Ala Phe Asp Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser Ser Ala
                  115 120
           <![CDATA[ <210> 139]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of OX40 agonist monoclonal antibody 011]]>
           <![CDATA[ <400> 139]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Pro Val Thr Pro Gly
          1 5 10 15
          Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
                      20 25 30
          Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ala Gly Gln Ser
                  35 40 45
          Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
              50 55 60
          Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
          65 70 75 80
          Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr
                          85 90 95
          Tyr Asn His Pro Thr Thr Phe Gly Gln Gly Thr Lys
                      100 105
           <![CDATA[ <210> 140]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of OX40 agonist monoclonal antibody 021]]>
           <![CDATA[ <400> 140]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Arg Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
                      20 25 30
          Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Lys Asp Arg Tyr Ile Thr Leu Pro Asn Ala Leu Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser
                  115 120
           <![CDATA[ <210> 141]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> OX40]]> Light chain variable region (VL) of agonist monoclonal antibody 021
           <![CDATA[ <400> 141]]>
          Asp Ile Gln Met Thr Gln Ser Pro Val Ser Leu Pro Val Thr Pro Gly
          1 5 10 15
          Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
                      20 25 30
          Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
                  35 40 45
          Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
              50 55 60
          Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
          65 70 75 80
          Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr
                          85 90 95
          Lys Ser Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys
                      100 105
           <![CDATA[ <210> 142]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of OX40 agonist monoclonal antibody 023]]>
           <![CDATA[ <400> 142]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr
                      20 25 30
          Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Met
              50 55 60
          Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
          65 70 75 80
          Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
                          85 90 95
          Arg Tyr Asp Asn Val Met Gly Leu Tyr Trp Phe Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 143]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> ]]>PRT
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of OX40 agonist monoclonal antibody 023]]>
           <![CDATA[ <400> 143]]>
          Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
                  35 40 45
          Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
          65 70 75 80
          Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
                          85 90 95
          Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 144]]>
           <![CDATA[ <211> 119]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> OX40 Agonist Monoclonal Antibody Heavy Chain Variable Region (VH)]]>
           <![CDATA[ <400> 144]]>
          Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
                  35 40 45
          Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe
              50 55 60
          Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Ser Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 145]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of OX40 agonist monoclonal antibody]]>
           <![CDATA[ <400> 145]]>
          Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Ser Leu Ser Ala Ser Leu Gly
          1 5 10 15
          Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
                      20 25 30
          Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
                  35 40 45
          Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
          65 70 75 80
          Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 146]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> OX40 Agonist Monoclonal Antibody Heavy Chain Variable Region (VH)]]>
           <![CDATA[ <400> 146]]>
          Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Lys Asp Tyr
                      20 25 30
          Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
                  35 40 45
          Gly Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn Gln Asn Phe
              50 55 60
          Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
          65 70 75 80
          Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Met Gly Tyr His Gly Pro His Leu Asp Phe Asp Val Trp Gly
                      100 105 110
          Ala Gly Thr Thr Val Thr Val Ser Pro
                  115 120
           <![CDATA[ <210> 147]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of OX40 agonist monoclonal antibody]]>
           <![CDATA[ <400> 147]]>
          Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Leu Gly
          1 5 10 15
          Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ala Ala
                      20 25 30
          Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
              50 55 60
          Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
          65 70 75 80
          Glu Asp Leu Thr Asp Tyr Phe Cys Gln Gln Tyr Ile Asn Tyr Pro Leu
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 148]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of humanized OX40 agonist monoclonal antibody]]>
           <![CDATA[ <400> 148]]>
          Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
          1 5 10 15
          Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
                      20 25 30
          Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
                  35 40 45
          Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
              50 55 60
          Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
                          85 90 95
          Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp
                      100 105 110
          Gly His Gly Thr Ser Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 149]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of humanized OX40 agonist monoclonal antibody]]>
           <![CDATA[ <400> 149]]>
          Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
                      20 25 30
          Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met
                  35 40 45
          Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
              50 55 60
          Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp
                      100 105 110
          Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 150]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of humanized OX40 agonist monoclonal antibody]]>
           <![CDATA[ <400> 150]]>
          Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Arg
          1 5 10 15
          Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
                      20 25 30
          Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
          65 70 75 80
          Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
                      100 105
           <![CDATA[ <210> 151]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain of humanized OX40 agonist monoclonal antibody]]> variable region (VL)
           <![CDATA[ <400> 151]]>
          Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Arg
          1 5 10 15
          Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
                      20 25 30
          Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
          65 70 75 80
          Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg
                          85 90 95
          Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
                      100 105
           <![CDATA[ <210> 152]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of humanized OX40 agonist monoclonal antibody]]>
           <![CDATA[ <400> 152]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Leu Ser Cys Glu Ser Asn Glu Tyr Glu Phe Pro Ser His
                      20 25 30
          Asp Met Ser Trp Val Arg Lys Thr Pro Glu Lys Arg Leu Glu Leu Val
                  35 40 45
          Ala Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met
              50 55 60
          Glu Arg Arg Phe Ile Ile Ser Arg Asp Asn Thr Lys Lys Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
                          85 90 95
          Ala Arg His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ala
                  115 120
           <![CDATA[ <210> 153]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain variable region (VH) of humanized OX40 agonist monoclonal antibody]]>
           <![CDATA[ <400> 153]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Tyr Glu Phe Pro Ser His
                      20 25 30
          Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val
                  35 40 45
          Ala Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met
              50 55 60
          Glu Arg Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Met Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 154]]>
           <![CDATA[ <211> 111]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of humanized OX40 agonist monoclonal antibody]]>
           <![CDATA[ <400> 154]]>
          Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser
                      20 25 30
          Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
                  35 40 45
          Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
              50 55 60
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
          65 70 75 80
          Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg
                          85 90 95
          Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
                      100 105 110
           <![CDATA[ <210> 155]]>
           <![CDATA[ <211> 111]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of humanized OX40 agonist monoclonal antibody]]>
           <![CDATA[ <400> 155]]>
          Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser
                      20 25 30
          Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
                  35 40 45
          Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
              50 55 60
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65 70 75 80
          Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg
                          85 90 95
          Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
                      100 105 110
           <![CDATA[ <210> 156]]>
           <![CDATA[ <211> 138]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> OX40 Agonist Monoclonal Antibody Heavy Chain Variable Region (VH)]]>
           <![CDATA[ <400> 156]]>
          Met Tyr Leu Gly Leu Asn Tyr Val Phe Ile Val Phe Leu Leu Asn Gly
          1 5 10 15
          Val Gln Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln
                      20 25 30
          Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
                  35 40 45
          Ser Asp Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu
              50 55 60
          Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr
          65 70 75 80
          Tyr Ala Glu Ser Val Asn Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
                          85 90 95
          Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
                      100 105 110
          Gly Ile Tyr Tyr Cys Thr Trp Gly Glu Val Phe Tyr Phe Asp Tyr Trp
                  115 120 125
          Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
              130 135
           <![CDATA[ <210> 157]]>
           <![CDATA[ <211> 126]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain variable region (VL) of OX40 agonist monoclonal antibody]]>
           <![CDATA[ <400> 157]]>
          Met Arg Pro Ser Ile Gln Phe Leu Gly Leu Leu Leu Phe Trp Leu His
          1 5 10 15
          Gly Ala Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
                      20 25 30
          Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ser Ser Gln Asp
                  35 40 45
          Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro
              50 55 60
          Arg Leu Leu Ile His Tyr Thr Ser Ser Thr Leu Gln Pro Gly Ile Pro Ser
          65 70 75 80
          Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser
                          85 90 95
          Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp
                      100 105 110
          Asn Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
                  115 120 125
           <![CDATA[ <210> 158]]>
           <![CDATA[ <211> 440]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy Chain Amino Acid Sequence of PD-1 Inhibitor Nivolumab]]>
           <![CDATA[ <400> 158]]>
          Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
          1 5 10 15
          Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
                      20 25 30
          Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
                      100 105 110
          Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
                  115 120 125
          Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
              130 135 140
          Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
          145 150 155 160
          Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
                          165 170 175
          Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys
                      180 185 190
          Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
                  195 200 205
          Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
              210 215 220
          Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
          225 230 235 240
          Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
                          245 250 255
          Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
                      260 265 270
          Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
                  275 280 285
          Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
              290 295 300
          Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
          305 310 315 320
          Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
                          325 330 335
          Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
                      340 345 350
          Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
                  355 360 365
          Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
              370 375 380
          Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
          385 390 395 400
          Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
                          405 410 415
          Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
                      420 425 430
          Ser Leu Ser Leu Ser Leu Gly Lys
                  435 440
           <![CDATA[ <210> 159]]>
           <![CDATA[ <211> 214]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain amino acid sequence of PD-1 inhibitor nivolumab]]>
           <![CDATA[ <400> 159]]>
          Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
                  35 40 45
          Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
          65 70 75 80
          Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
                      100 105 110
          Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
                  115 120 125
          Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
              130 135 140
          Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
          145 150 155 160
          Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
                          165 170 175
          Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
                      180 185 190
          Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
                  195 200 205
          Phe Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> ]]>160
           <![CDATA[ <211> 113]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain variable region (VH) of PD-1 inhibitor nivolumab]]>
           <![CDATA[ <400> 160]]>
          Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
          1 5 10 15
          Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
                      20 25 30
          Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
                      100 105 110
          Ser
           <![CDATA[ <210> 161]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of light chain variable region (VL) of PD-1 inhibitor nivolumab]]>
           <![CDATA[ <400> 161]]>
          Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
                  35 40 45
          Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
          65 70 75 80
          Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
                      100 105
           <![CDATA[ <210> 162]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR1 of PD-1 inhibitor nivolumab]]>
           <![CDATA[ <400> 162]]>
          Asn Ser Gly Met His
          1 5
           <![CDATA[ <210> 163]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR2 of PD-1 inhibitor nivolumab]]>
           <![CDATA[ <400> 163]]>
          Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 164]]>
           <![CDATA[ <211> 4]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR3 of PD-1 inhibitor nivolumab]]>
           <![CDATA[ <400> 164]]>
          Asn Asp Asp Tyr
          1               
           <![CDATA[ <210> 165]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 amino acid sequence of PD-1 inhibitor nivolumab]]>
           <![CDATA[ <400> 165]]>
          Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala
          1 5 10
           <![CDATA[ <210> 166]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 amino acid sequence of PD-1 inhibitor nivolumab]]>
           <![CDATA[ <400> 166]]>
          Asp Ala Ser Asn Arg Ala Thr
          1 5
           <![CDATA[ <210> 167]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 amino acid sequence of PD-1 inhibitor nivolumab]]>
           <![CDATA[ <400> 167]]>
          Gln Gln Ser Ser Asn Trp Pro Arg Thr
          1 5
           <![CDATA[ <210> 168]]>
           <![CDATA[ <211> 447]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy Chain Amino Acid Sequence of PD-1 Inhibitor Pembrolizumab]]>
           <![CDATA[ <400> 168]]>
          Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
                      20 25 30
          Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
              50 55 60
          Lys Asn Arg Val Thr Leu Thr Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr
          65 70 75 80
          Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
                  115 120 125
          Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
              130 135 140
          Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
          145 150 155 160
          Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
                          165 170 175
          Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
                      180 185 190
          Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
                  195 200 205
          Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
              210 215 220
          Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val
          225 230 235 240
          Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
                          245 250 255
          Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
                      260 265 270
          Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
                  275 280 285
          Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
              290 295 300
          Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
          305 310 315 320
          Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
                          325 330 335
          Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
                      340 345 350
          Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
                  355 360 365
          Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
              370 375 380
          Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
          385 390 395 400
          Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
                          405 410 415
          Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
                      420 425 430
          His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
                  435 440 445
           <![CDATA[ <210> 169]]>
           <![CDATA[ <211> 218]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain amino acid sequence of PD-1 inhibitor pembrolizumab]]>
           <![CDATA[ <400> 169]]>
          Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser
                      20 25 30
          Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
                  35 40 45
          Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala
              50 55 60
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65 70 75 80
          Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg
                          85 90 95
          Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
                      100 105 110
          Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
                  115 120 125
          Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr
              130 135 140
          Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
          145 150 155 160
          Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
                          165 170 175
          Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
                      180 185 190
          His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
                  195 200 205
          Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
              210 215
           <![CDATA[ <210> 170]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain variable region (VH) of PD-1 inhibitor pembrolizumab]]>
           <![CDATA[ <400> 170]]>
          Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
                      20 25 30
          Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
              50 55 60
          Lys Asn Arg Val Thr Leu Thr Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr
          65 70 75 80
          Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 171]]>
           <![CDATA[ <211> 111]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of light chain variable region (VL) of PD-1 inhibitor pembrolizumab]]>
           <![CDATA[ <400> 171]]>
          Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser
                      20 25 30
          Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
                  35 40 45
          Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala
              50 55 60
          Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
          65 70 75 80
          Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg
                          85 90 95
          Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
                      100 105 110
           <![CDATA[ <210> 172]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR1 of PD-1 inhibitor pembrolizumab]]>
           <![CDATA[ <400> 172]]>
          Asn Tyr Tyr Met Tyr
          1 5
           <![CDATA[ <210> 173]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR2 of PD-1 inhibitor pembrolizumab]]>
           <![CDATA[ <400> 173]]>
          Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys
          1 5 10 15
           <![CDATA[ <210> 174]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR3 of PD-1 inhibitor pembrolizumab]]>
           <![CDATA[ <400> 174]]>
          Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 175]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 amino acid sequence of PD-1 inhibitor pembrolizumab]]>
           <![CDATA[ <400> 175]]>
          Arg Ala Ser Lys Gly Val Ser Thr Ser Gly Tyr Ser Tyr Leu His
          1 5 10 15
           <![CDATA[ <210> 176]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 amino acid sequence of PD-1 inhibitor pembrolizumab]]>
           <![CDATA[ <400> 176]]>
          Leu Ala Ser Tyr Leu Glu Ser
          1 5
           <![CDATA[ <210> 177]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 amino acid sequence of PD-1 inhibitor pembrolizumab]]>
           <![CDATA[ <400> 177]]>
          Gln His Ser Arg Asp Leu Pro Leu Thr
          1 5
           <![CDATA[ <210> 178]]>
           <![CDATA[ <211> 451]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain amino acid sequence of PD-L1 inhibitor durvalumab]]>
           <![CDATA[ <400> 178]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Glu Gly Gly Trp Phe Gly Glu Leu Ala Phe Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
                  115 120 125
          Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
              130 135 140
          Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
          145 150 155 160
          Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
                          165 170 175
          Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
                      180 185 190
          Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
                  195 200 205
          Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
              210 215 220
          Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly
          225 230 235 240
          Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
                          245 250 255
          Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
                      260 265 270
          Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
                  275 280 285
          His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
              290 295 300
          Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
          305 310 315 320
          Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile
                          325 330 335
          Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
                      340 345 350
          Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
                  355 360 365
          Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
              370 375 380
          Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
          385 390 395 400
          Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
                          405 410 415
          Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
                      420 425 430
          His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
                  435 440 445
          Pro Gly Lys
              450
           <![CDATA[ <210> 179]]>
           <![CDATA[ <211> 26]]>5
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain amino acid sequence of PD-L1 inhibitor durvalumab]]>
           <![CDATA[ <400> 179]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Asn Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
              50 55 60
          Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Val Ser
          65 70 75 80
          Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
                          85 90 95
          Leu Leu Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
                      100 105 110
          Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
                  115 120 125
          Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser
              130 135 140
          Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
          145 150 155 160
          Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
                          165 170 175
          Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr Pro
                      180 185 190
          Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
                  195 200 205
          Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
              210 215 220
          Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
          225 230 235 240
          Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
                          245 250 255
          Thr Lys Ser Phe Asn Arg Gly Glu Cys
                      260 265
           <![CDATA[ <210> 180]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain variable region (VH) of PD-L1 inhibitor durvalumab]]>
           <![CDATA[ <400> 180]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Glu Gly Gly Trp Phe Gly Glu Leu Ala Phe Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 181]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of light chain variable region (VL) of PD-L1 inhibitor durvalumab]]>
           <![CDATA[ <400> 181]]>
          Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser
                      20 25 30
          Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
                  35 40 45
          Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
              50 55 60
          Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
          65 70 75 80
          Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Leu Pro
                          85 90 95
          Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
                      100 105
           <![CDATA[ <210> 182]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR1 of PD-L1 inhibitor durvalumab]]>
           <![CDATA[ <400> 182]]>
          Arg Tyr Trp Met Ser
          1 5
           <![CDATA[ <210> 183]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR2 of PD-L1 inhibitor durvalumab]]>
           <![CDATA[ <400> 183]]>
          Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 184]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR3 of PD-L1 inhibitor durvalumab]]>
           <![CDATA[ <400> 184]]>
          Glu Gly Gly Trp Phe Gly Glu Leu Ala Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 185]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 amino acid sequence of PD-L1 inhibitor durvalumab]]>
           <![CDATA[ <400> 185]]>
          Arg Ala Ser Gln Arg Val Ser Ser Ser Tyr Leu Ala
          1 5 10
           <![CDATA[ <210> 186]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 amino acid sequence of PD-L1 inhibitor durvalumab]]>
           <![CDATA[ <400> 186]]>
          Asp Ala Ser Ser Arg Ala Thr
          1 5
           <![CDATA[ <210> 187]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 amino acid sequence of PD-L1 inhibitor durvalumab]]>
           <![CDATA[ <400> 187]]>
          Gln Gln Tyr Gly Ser Leu Pro Trp Thr
          1 5
           <![CDATA[ <210> 188]]>
           <![CDATA[ <211> 450]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> PD-L1 Inhibitor Avi]]> Heavy Chain Amino Acid Sequence of Lumumab
           <![CDATA[ <400> 188]]>
          Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
                      20 25 30
          Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
                  115 120 125
          Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
              130 135 140
          Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
          145 150 155 160
          Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
                          165 170 175
          Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
                      180 185 190
          Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
                  195 200 205
          Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
              210 215 220
          Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
          225 230 235 240
          Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
                          245 250 255
          Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
                      260 265 270
          Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
                  275 280 285
          Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
              290 295 300
          Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
          305 310 315 320
          Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
                          325 330 335
          Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
                      340 345 350
          Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
                  355 360 365
          Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
              370 375 380
          Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
          385 390 395 400
          Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
                          405 410 415
          Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
                      420 425 430
          Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                  435 440 445
          Gly Lys
              450
           <![CDATA[ <210> 189]]>
           <![CDATA[ <211> 216]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain amino acid sequence of PD-L1 inhibitor avelumab]]>
           <![CDATA[ <400> 189]]>
          Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
          1 5 10 15
          Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
                      20 25 30
          Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
                  35 40 45
          Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
              50 55 60
          Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
          65 70 75 80
          Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser
                          85 90 95
          Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln
                      100 105 110
          Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
                  115 120 125
          Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
              130 135 140
          Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys
          145 150 155 160
          Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr
                          165 170 175
          Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
                      180 185 190
          Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
                  195 200 205
          Thr Val Ala Pro Thr Glu Cys Ser
              210 215
           <![CDATA[ <210> 190]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain variable region (VH) of PD-L1 inhibitor avelumab]]>
           <![CDATA[ <400> 190]]>
          Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
                      20 25 30
          Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 191]]>
           <![CDATA[ <211> 110]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of light chain variable region (VL) of PD-L1 inhibitor avelumab]]>
           <![CDATA[ <400> 191]]>
          Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
          1 5 10 15
          Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
                      20 25 30
          Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
                  35 40 45
          Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
              50 55 60
          Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
          65 70 75 80
          Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser
                          85 90 95
          Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu
                      100 105 110
           <![CDATA[ <210> 192]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR1 of PD-L1 inhibitor avelumab]]>
           <![CDATA[ <400> 192]]>
          Ser Tyr Ile Met Met
          1 5
           <![CDATA[ <210> 193]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR2 of PD-L1 inhibitor avelumab]]>
           <![CDATA[ <400> 193]]>
          Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 194]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR3 of PD-L1 inhibitor avelumab]]>
           <![CDATA[ <400> 194]]>
          Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr
          1 5 10
           <![CDATA[ <210> 195]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 amino acid sequence of PD-L1 inhibitor avelumab]]>
           <![CDATA[ <400> 195]]>
          Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser
          1 5 10
           <![CDATA[ <210> 196]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 amino acid sequence of PD-L1 inhibitor avelumab]]>
           <![CDATA[ <400> 196]]>
          Asp Val Ser Asn Arg Pro Ser
          1 5
           <![CDATA[ <210> 197]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 amino acid sequence of PD-L1 inhibitor avelumab]]>
           <![CDATA[ <400> 197]]>
          Ser Ser Tyr Thr Ser Ser Ser Thr Arg Val
          1 5 10
           <![CDATA[ <210> 198]]>
           <![CDATA[ <211> 448]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy Chain Amino Acid Sequence of PD-L1 Inhibitor Atezolizumab]]>
           <![CDATA[ <400> 198]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
                      20 25 30
          Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
                      100 105 110
          Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
                  115 120 125
          Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
              130 135 140
          Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
          145 150 155 160
          Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
                          165 170 175
          Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser
                      180 185 190
          Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
                  195 200 205
          Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
              210 215 220
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
          225 230 235 240
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                          245 250 255
          Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
                      260 265 270
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
                  275 280 285
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
              290 295 300
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
          305 310 315 320
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                          325 330 335
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                      340 345 350
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
                  355 360 365
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
              370 375 380
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
          385 390 395 400
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                          405 410 415
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                      420 425 430
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                  435 440 445
           <![CDATA[ <210> 199]]>
           <![CDATA[ <211> 214]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain amino acid sequence of PD-L1 inhibitor atezolizumab]]>
           <![CDATA[ <400> 199]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
                      20 25 30
          Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
                      100 105 110
          Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
                  115 120 125
          Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
              130 135 140
          Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
          145 150 155 160
          Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
                          165 170 175
          Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
                      180 185 190
          Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
                  195 200 205
          Phe Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 200]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain variable region (VH) of PD-L1 inhibitor atezolizumab]]>
           <![CDATA[ <400> 200]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
                      20 25 30
          Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
                      100 105 110
          Leu Val Thr Val Ser Ala
                  115
           <![CDATA[ <210> 201]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of light chain variable region (VL) of PD-L1 inhibitor atezolizumab]]>
           <![CDATA[ <400> 201]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
                      20 25 30
          Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
                      100 105
           <![CDATA[ <210> 202]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR1 of PD-L1 inhibitor atezolizumab]]>
           <![CDATA[ <400> 202]]>
          Gly Phe Thr Phe Ser Asp Ser Trp Ile His
          1 5 10
           <![CDATA[ <210> 203]]>
           <![CDATA[ <211> 18]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> PD-L1 Inhibitor Atezolizumab Heavy Chain C]]>DR2 Amino Acid Sequence
           <![CDATA[ <400> 203]]>
          Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
          1 5 10 15
          Lys Gly
           <![CDATA[ <210> 204]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR3 of PD-L1 inhibitor atezolizumab]]>
           <![CDATA[ <400> 204]]>
          Arg His Trp Pro Gly Gly Phe Asp Tyr
          1 5
           <![CDATA[ <210> 205]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 amino acid sequence of PD-L1 inhibitor atezolizumab]]>
           <![CDATA[ <400> 205]]>
          Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala
          1 5 10
           <![CDATA[ <210> 206]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 amino acid sequence of PD-L1 inhibitor atezolizumab]]>
           <![CDATA[ <400> 206]]>
          Ser Ala Ser Phe Leu Tyr Ser
          1 5
           <![CDATA[ <210> 207]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> PD-]]>L1 Inhibitor Atezolizumab Light Chain CDR3 Amino Acid Sequence
           <![CDATA[ <400> 207]]>
          Gln Gln Tyr Leu Tyr His Pro Ala Thr
          1 5
           <![CDATA[ <210> 208]]>
           <![CDATA[ <211> 225]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy chain amino acid sequence of CTLA-4 inhibitor ipilimumab]]>
           <![CDATA[ <400> 208]]>
          Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
                      20 25 30
          Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys
                          85 90 95
          Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr
                      100 105 110
          Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
                  115 120 125
          Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
              130 135 140
          Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
          145 150 155 160
          Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
                          165 170 175
          Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser
                      180 185 190
          Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
                  195 200 205
          Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
              210 215 220
          His
          225
           <![CDATA[ <210> 209]]>
           <![CDATA[ <211> 215]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain amino acid sequence of CTLA-4 inhibitor ipilimumab]]>
           <![CDATA[ <400>]]> 209
          Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser
                      20 25 30
          Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
                  35 40 45
          Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
              50 55 60
          Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
          65 70 75 80
          Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
                          85 90 95
          Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
                      100 105 110
          Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
                  115 120 125
          Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
              130 135 140
          Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
          145 150 155 160
          Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
                          165 170 175
          Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
                      180 185 190
          Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
                  195 200 205
          Ser Phe Asn Arg Gly Glu Cys
              210 215
           <![CDATA[ <210> 210]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain variable region (VH) of CTLA-4 inhibitor ipilimumab]]>
           <![CDATA[ <400> 210]]>
          Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
                      20 25 30
          Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys
                          85 90 95
          Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr
                      100 105 110
          Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 211]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of light chain variable region (VL) of CTLA-4 inhibitor ipilimumab]]>
           <![CDATA[ <400> 211]]>
          Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser
                      20 25 30
          Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
                  35 40 45
          Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
              50 55 60
          Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
          65 70 75 80
          Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
                          85 90 95
          Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
                      100 105
           <![CDATA[ <210> 212]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> CTLA-4 Inhibitor I]]> Heavy Chain CDR1 Amino Acid Sequence of Pilimumab
           <![CDATA[ <400> 212]]>
          Gly Phe Thr Phe Ser Ser Tyr Thr
          1 5
           <![CDATA[ <210> 213]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR2 of CTLA-4 inhibitor ipilimumab]]>
           <![CDATA[ <400> 213]]>
          Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys
          1 5 10
           <![CDATA[ <210> 214]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR3 of CTLA-4 inhibitor ipilimumab]]>
           <![CDATA[ <400> 214]]>
          Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 215]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 amino acid sequence of CTLA-4 inhibitor ipilimumab]]>
           <![CDATA[ <400> 215]]>
          Gln Ser Val Gly Ser Ser Tyr
          1 5
           <![CDATA[ <210> 216]]>
           <![CDATA[ <211> 3]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 amino acid sequence of CTLA-4 inhibitor ipilimumab]]>
           <![CDATA[ <400> 216]]>
          Gly Ala Phe
          1           
           <![CDATA[ <210> 217]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 amino acid sequence of CTLA-4 inhibitor ipilimumab]]>
           <![CDATA[ <400> 217]]>
          Gln Gln Tyr Gly Ser Ser Pro Trp Thr
          1 5
           <![CDATA[ <210> 218]]>
           <![CDATA[ <211> 451]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy Chain Amino Acid Sequence of CTLA-4 Inhibitor Tremelimumab]]>
           <![CDATA[ <400> 218]]>
          Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
                      20 25 30
          Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Asp Pro Arg Gly Ala Thr Leu Tyr Tyr Tyr Tyr Tyr Tyr Gly Met
                      100 105 110
          Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
                  115 120 125
          Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
              130 135 140
          Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
          145 150 155 160
          Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
                          165 170 175
          Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
                      180 185 190
          Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys
                  195 200 205
          Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu
              210 215 220
          Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
          225 230 235 240
          Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
                          245 250 255
          Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
                      260 265 270
          Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
                  275 280 285
          His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
              290 295 300
          Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly
          305 310 315 320
          Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile
                          325 330 335
          Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
                      340 345 350
          Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
                  355 360 365
          Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
              370 375 380
          Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
          385 390 395 400
          Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
                          405 410 415
          Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
                      420 425 430
          His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
                  435 440 445
          Pro Gly Lys
              450
           <![CDATA[ <210> 219]]>
           <![CDATA[ <211> 214]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain amino acid sequence of CTLA-4 inhibitor tremelimumab]]>
           <![CDATA[ <400> 219]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Tyr
                      20 25 30
          Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Ala Ala Ser Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Phe
                          85 90 95
          Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
                      100 105 110
          Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
                  115 120 125
          Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
              130 135 140
          Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
          145 150 155 160
          Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
                          165 170 175
          Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
                      180 185 190
          Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
                  195 200 205
          Phe Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 220]]>
           <![CDATA[ <211> 167]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain variable region (VH) of CTLA-4 inhibitor tremelimumab]]>
           <![CDATA[ <400> 220]]>
          Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser
          1 5 10 15
          Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro
                      20 25 30
          Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn
                  35 40 45
          Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
              50 55 60
          Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
          65 70 75 80
          Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Pro Arg Gly Ala Thr Leu
                          85 90 95
          Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val
                      100 105 110
          Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
                  115 120 125
          Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
              130 135 140
          Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
          145 150 155 160
          Ala Leu Thr Ser Gly Val His
                          165
           <![CDATA[ <210> 221]]>
           <![CDATA[ <211> 139]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of light chain variable region (VL) of CTLA-4 inhibitor tremelimumab]]>
           <![CDATA[ <400> 221]]>
          Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
          1 5 10 15
          Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asp Trp Tyr Gln Gln Lys
                      20 25 30
          Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Ser Leu Gln
                  35 40 45
          Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
              50 55 60
          Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
          65 70 75 80
          Cys Gln Gln Tyr Tyr Ser Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys
                          85 90 95
          Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
                      100 105 110
          Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
                  115 120 125
          Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val
              130 135
           <![CDATA[ <210> 222]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR1 of CTLA-4 inhibitor tremelimumab]]>
           <![CDATA[ <400> 222]]>
          Gly Phe Thr Phe Ser Ser Tyr Gly Met His
          1 5 10
           <![CDATA[ <210> 223]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR2 of CTLA-4 inhibitor tremelimumab]]>
           <![CDATA[ <400> 223]]>
          Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
          1 5 10 15
           <![CDATA[ <210> 224]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR3 of CTLA-4 inhibitor tremelimumab]]>
           <![CDATA[ <400> 224]]>
          Asp Pro Arg Gly Ala Thr Leu Tyr Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val
          1 5 10 15
           <![CDATA[ <210> 225]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 amino acid sequence of CTLA-4 inhibitor tremelimumab]]>
           <![CDATA[ <400> 225]]>
          Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asp
          1 5 10
           <![CDATA[ <210> 226]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 amino acid sequence of CTLA-4 inhibitor tremezumab]]>
           <![CDATA[ <400> 226]]>
          Ala Ala Ser Ser Leu Gln Ser
          1 5
           <![CDATA[ <210> 227]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 amino acid sequence of CTLA-4 inhibitor tremelimumab]]>
           <![CDATA[ <400> 227]]>
          Gln Gln Tyr Tyr Ser Thr Pro Phe Thr
          1 5
           <![CDATA[ <210> 228]]>
           <![CDATA[ <211> 448]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy Chain Amino Acid Sequence of CTLA-4 Inhibitor Zefelizumab]]>
           <![CDATA[ <400> 228]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
                      20 25 30
          Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Val Gly Leu Met Gly Pro Phe Asp Ile Trp Gly Gln Gly Thr
                      100 105 110
          Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
                  115 120 125
          Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
              130 135 140
          Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
          145 150 155 160
          Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
                          165 170 175
          Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser
                      180 185 190
          Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
                  195 200 205
          Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
              210 215 220
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
          225 230 235 240
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                          245 250 255
          Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
                      260 265 270
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
                  275 280 285
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
              290 295 300
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
          305 310 315 320
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                          325 330 335
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                      340 345 350
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
                  355 360 365
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
              370 375 380
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
          385 390 395 400
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                          405 410 415
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                      420 425 430
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                  435 440 445
           <![CDATA[ <210> 229]]>
           <![CDATA[ <211> 214]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain amino acid sequence of CTLA-4 inhibitor Zefelizumab]]>
           <![CDATA[ <400> 229]]>
          Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr
                      20 25 30
          Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
                  35 40 45
          Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro
          65 70 75 80
          Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Trp
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
                      100 105 110
          Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
                  115 120 125
          Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
              130 135 140
          Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
          145 150 155 160
          Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
                          165 170 175
          Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
                      180 185 190
          Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
                  195 200 205
          Phe Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 230]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain variable region (VH) of CTLA-4 inhibitor Zefelizumab]]>
           <![CDATA[ <400> 230]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
                      20 25 30
          Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Val Gly Leu Met Gly Pro Phe Asp Ile Trp Gly Gln Gly Thr
                      100 105 110
          Met Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 231]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of light chain variable region (VL) of CTLA-4 inhibitor Zefelizumab]]>
           <![CDATA[ <400> 231]]>
          Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr
                      20 25 30
          Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
                  35 40 45
          Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro
          65 70 75 80
          Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Trp
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
                      100 105
           <![CDATA[ <210> 232]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR1 of CTLA-4 inhibitor Zefelizumab]]>
           <![CDATA[ <400> 232]]>
          Gly Phe Thr Phe Ser Ser Tyr Ser
          1 5
           <![CDATA[ <210> 233]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR2 of CTLA-4 inhibitor Zefelizumab]]>
           <![CDATA[ <400> 233]]>
          Ile Ser Ser Ser Ser Ser Tyr Ile
          1 5
           <![CDATA[ <210> 234]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Amino acid sequence of heavy chain CDR3 of CTLA-4 inhibitor Zefelizumab]]>
           <![CDATA[ <400> 234]]>
          Ala Arg Val Gly Leu Met Gly Pro Phe Asp Ile
          1 5 10
           <![CDATA[ <210> 235]]>
           <![CDATA[ <211> 6]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR1 amino acid sequence of CTLA-4 inhibitor Zefelizumab]]>
           <![CDATA[ <400> 235]]>
          Gln Ser Val Ser Arg Tyr
          1 5
           <![CDATA[ <210> 236]]>
           <![CDATA[ <211> 3]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR2 amino acid sequence of CTLA-4 inhibitor Zefelizumab]]>
           <![CDATA[ <400> 236]]>
          Gly Ala Ser
          1           
           <![CDATA[ <210> 237]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Light chain CDR3 amino acid sequence of CTLA-4 inhibitor Zefelizumab]]>
           <![CDATA[ <400> 237]]>
          Gln Gln Tyr Gly Ser Ser Pro Trp Thr
          1 5
           <![CDATA[ <210> 238]]>
           <![CDATA[ <211> 49]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> target PD-1 sequence]]>
           <![CDATA[ <400> 238]]>
          ttctccccag ccctgctcgt ggtgaccgaa ggggacaacg ccaccttca 49
           <![CDATA[ <210> 239]]>
           <![CDATA[ <211> 49]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> target PD-1 sequence]]>
           <![CDATA[ <400> 239]]>
          tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagaga 49
           <![CDATA[ <210> 240]]>
           <![CDATA[ <211> 530]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Repeat PD-1-Left]]>
           <![CDATA[ <400> 240]]>
          Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
          1 5 10 15
          Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
                      20 25 30
          His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
                  35 40 45
          Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
              50 55 60
          Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
          65 70 75 80
          Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
                          85 90 95
          Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
                      100 105 110
          Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
                  115 120 125
          Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
              130 135 140
          Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
          145 150 155 160
          Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
                          165 170 175
          Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
                      180 185 190
          Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
                  195 200 205
          Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
              210 215 220
          Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
          225 230 235 240
          Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
                          245 250 255
          Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
                      260 265 270
          Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
                  275 280 285
          Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
              290 295 300
          His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
          305 310 315 320
          Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
                          325 330 335
          Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
                      340 345 350
          Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
                  355 360 365
          Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
              370 375 380
          Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
          385 390 395 400
          Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
                          405 410 415
          Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
                      420 425 430
          Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
                  435 440 445
          Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
              450 455 460
          Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
          465 470 475 480
          Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
                          485 490 495
          Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
                      500 505 510
          Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
                  515 520 525
          Leu Glu
              530
           <![CDATA[ <210> 241]]>
           <![CDATA[ <211> 529]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Repeat PD-1-Right]]>
           <![CDATA[ <400> 241]]>
          Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
          1 5 10 15
          Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
                      20 25 30
          His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
                  35 40 45
          Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys
              50 55 60
          Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
          65 70 75 80
          Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
                          85 90 95
          Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
                      100 105 110
          Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
                  115 120 125
          Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
              130 135 140
          Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
          145 150 155 160
          Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
                          165 170 175
          Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
                      180 185 190
          Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
                  195 200 205
          Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
              210 215 220
          Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
          225 230 235 240
          Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala
                          245 250 255
          Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
                      260 265 270
          Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Lys Gln
                  275 280 285
          Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
              290 295 300
          Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
          305 310 315 320
          Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
                          325 330 335
          Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly
                      340 345 350
          Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
                  355 360 365
          Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser
              370 375 380
          Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
          385 390 395 400
          Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile
                          405 410 415
          Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
                      420 425 430
          Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val
                  435 440 445
          Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
              450 455 460
          Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
          465 470 475 480
          Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr
                          485 490 495
          Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
                      500 505 510
          Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala Leu
                  515 520 525
          Glu
           <![CDATA[ <210> 242]]>
           <![CDATA[ <211> 530]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Repeat PD-1-Left]]>
           <![CDATA[ <400> 242]]>
          Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
          1 5 10 15
          Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala
                      20 25 30
          His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
                  35 40 45
          Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
              50 55 60
          Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
          65 70 75 80
          Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
                          85 90 95
          Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
                      100 105 110
          Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
                  115 120 125
          Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
              130 135 140
          Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
          145 150 155 160
          Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
                          165 170 175
          Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
                      180 185 190
          Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
                  195 200 205
          Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
              210 215 220
          Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
          225 230 235 240
          Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala
                          245 250 255
          Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
                      260 265 270
          Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
                  275 280 285
          Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
              290 295 300
          His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
          305 310 315 320
          Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
                          325 330 335
          Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
                      340 345 350
          Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
                  355 360 365
          Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
              370 375 380
          Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
          385 390 395 400
          Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
                          405 410 415
          Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
                      420 425 430
          Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
                  435 440 445
          Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
              450 455 460
          Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
          465 470 475 480
          Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
                          485 490 495
          Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
                      500 505 510
          Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
                  515 520 525
          Leu Glu
              530
           <![CDATA[ <210> 243]]>
           <![CDATA[ <211> 529]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Heavy]]> Heavy PD-1-Right
           <![CDATA[ <400> 243]]>
          Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
          1 5 10 15
          Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
                      20 25 30
          His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
                  35 40 45
          Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
              50 55 60
          Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
          65 70 75 80
          Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
                          85 90 95
          Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
                      100 105 110
          Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
                  115 120 125
          Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
              130 135 140
          Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
          145 150 155 160
          Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
                          165 170 175
          Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
                      180 185 190
          Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
                  195 200 205
          Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
              210 215 220
          Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
          225 230 235 240
          Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
                          245 250 255
          Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
                      260 265 270
          Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
                  275 280 285
          Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
              290 295 300
          His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
          305 310 315 320
          Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
                          325 330 335
          Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
                      340 345 350
          Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
                  355 360 365
          Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
              370 375 380
          Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
          385 390 395 400
          Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
                          405 410 415
          Ile Ala Ser Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
                      420 425 430
          Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val
                  435 440 445
          Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
              450 455 460
          Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
          465 470 475 480
          Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr
                          485 490 495
          Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
                      500 505 510
          Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala Leu
                  515 520 525
          Glu
           <![CDATA[ <210> 244]]>
           <![CDATA[ <211> 2814]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> PD-1-Left TALEN]]>
           <![CDATA[ <400> 244]]>
          atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
          gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
          aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
          acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
          aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
          ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
          agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
          gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
          ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 540
          acggtccagc ggctgttgcc ggtgctgtgc caggccacg gcttgacccc ggagcaggtg 600
          gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 660
          ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 720
          ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
          cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 840
          ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 900
          caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 960
          ctgttgccgg tgctgtgcca ggccacggc ttgaccccgg agcaggtggt ggccatcgcc 1020
          agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
          caggccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga tggcggcaag 1140
          caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
          ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct ggagacggtg 1260
          caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1320
          atcgccagca ataatggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
          ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag ccacgatggc 1440
          ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
          ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 1560
          acggtccagc ggctgttgcc ggtgctgtgc caggccacg gcttgacccc ggagcaggtg 1620
          gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 1680
          ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1740
          ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
          cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 1860
          ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
          caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1980
          ctgttgccgg tgctgtgcca ggccacggc ttgaccccctc agcaggtggt ggccatcgcc 2040
          agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
          ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
          cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
          aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
          gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
          aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
          aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
          gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
          aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
          gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
          aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
          tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
          gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
           <![CDATA[ <210> 245]]>
           <![CDATA[ <211> 2829]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> PD-1-right TALEN]]>
           <![CDATA[ <400> 245]]>
          atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
          gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
          cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
          ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
          ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
          gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
          acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
          attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
          acgggtgccc cgctcaactt gaccccccag caagtcgtcg caatcgccag caataacgga 540
          gggaagcaag ccctcgaaac cgtgcagcgg ttgcttcctg tgctctgcca ggcccacggc 600
          cttacccctg agcaggtggt ggccatcgca agtaacattg gaggaaagca agccttggag 660
          acagtgcagg ccctgttgcc cgtgctgtgc caggcacacg gcctcacacc agagcaggtc 720
          gtggccattg cctccaacat cggggggaaa caggctctgg agaccgtcca ggccctgctg 780
          cccgtcctct gtcaagctca cggcctgact ccccaacaag tggtcgccat cgcctctaat 840
          aacggcggga agcaggcact ggaaacagtg cagagactgc tccctgtgct ttgccaagct 900
          catgggttga ccccccaaca ggtcgtcgct attgcctcaa acaacgggggg caagcaggcc 960
          cttgagactg tgcagaggct gttgccagtg ctgtgtcagg ctcacgggct cactccacaa 1020
          caggtggtcg caattgccag caacggcggc ggaaagcaag ctcttgaaac cgtgcaacgc 1080
          ctcctgcccg tgctctgtca ggctcatggc ctgacaccac aacaagtcgt ggccatcgcc 1140
          agtaataatg gcgggaaaca ggctcttgag accgtccaga ggctgctccc agtgctctgc 1200
          caggcacacg ggctgacccc ccagcaggtg gtggctatcg ccagcaataa tgggggcaag 1260
          caggccctgg aaacagtcca gcgcctgctg ccagtgcttt gccaggctca cgggctcact 1320
          cccgaacagg tcgtggcaat cgcctccaac ggagggaagc aggctctgga gaccgtgcag 1380
          agactgctgc ccgtcttgtg ccaggcccac ggactcacac ctcagcaggt cgtcgccatt 1440
          gcctctaaca acgggggcaa acaagccctg gagacagtgc agcggctgtt gcctgtgttg 1500
          tgccaagccc acggcttgac tcctcaacaa gtggtcgcca tcgcctcaaa tggcggcgga 1560
          aaacaagctc tggagacagt gcagaggttg ctgcccgtcc tctgccaagc ccacggcctg 1620
          actccccaac aggtcgtcgc cattgccagc aacggcggag gaaagcaggc tctcgaaact 1680
          gtgcagcggc tgcttcctgt gctgtgtcag gctcatgggc tgacccccca gcaagtggtg 1740
          gctattgcct ctaacaatgg aggcaagcaa gcccttgaga cagtccagag gctgttgcca 1800
          gtgctgtgcc aggcccacgg gctcacaccc cagcaggtgg tcgccatcgc cagtaacggc 1860
          gggggcaaac aggcattgga aaccgtccag cgcctgcttc cagtgctctg ccaggcacac 1920
          ggactgacac ccgaacaggt ggtggccatt gcatcccatg atgggggcaa gcaggccctg 1980
          gagaccgtgc agagactcct gccagtgttg tgccaagctc acggcctcac ccctcagcaa 2040
          gtcgtggcca tcgcctcaaa cggggggggc cggcctgcac tggagagcat tgttgcccag 2100
          ttatctcgcc ctgatccggc gttggccgcg ttgaccaacg accacctcgt cgccttggcc 2160
          tgcctcggcg ggcgtcctgc gctggatgca gtgaaaaagg gattggggga tcctatcagc 2220
          cgttcccagc tggtgaagtc cgagctggag gagaagaaat ccgagttgag gcacaagctg 2280
          aagtacgtgc cccacgagta catcgagctg atcgagatcg cccggaacag cacccaggac 2340
          cgtatcctgg agatgaaggt gatggagttc ttcatgaagg tgtacggcta caggggcaag 2400
          cacctgggcg gctccaggaa gcccgacggc gccatctaca ccgtgggctc ccccatcgac 2460
          tacggcgtga tcgtggacac caaggcctac tccggcggct acaacctgcc catcggccag 2520
          gccgacgaaa tgcagaggta cgtggaggag aaccagacca ggaacaagca catcaaccccc 2580
          aacgagtggt ggaaggtgta cccctccagc gtgaccgagt tcaagttcct gttcgtgtcc 2640
          ggccacttca agggcaacta caaggcccag ctgaccaggc tgaaccacat caccaactgc 2700
          aacggcgccg tgctgtccgt ggaggagctc ctgatcggcg gcgagatgat caaggccggc 2760
          accctgaccc tggaggaggt gaggaggaag ttcaacaacg gcgagatcaa cttcgcggcc 2820
          gactgataa 2829
           <![CDATA[ <210> 246]]>
           <![CDATA[ <211> 2814]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> PD-1-Left TALEN]]>
           <![CDATA[ <400> 246]]>
          atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
          gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
          aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
          acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
          aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
          ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
          agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
          gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
          ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 540
          acggtgcagg cgctgttgcc ggtgctgtgc caggccacg gcttgacccc ggagcaggtg 600
          gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 660
          ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 720
          gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
          cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 840
          ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 900
          caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 960
          ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1020
          agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
          caggccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaataa tggtggcaag 1140
          caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
          ccccagcagg tggtggccat cgccagcaat ggcggtggca agcaggcgct ggagacggtc 1260
          cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1320
          atcgccagca ataatggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
          ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caataatggt 1440
          ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
          ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 1560
          acggtccagc ggctgttgcc ggtgctgtgc caggccacg gcttgacccc ccagcaggtg 1620
          gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 1680
          ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 1740
          gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
          cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 1860
          ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
          caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac ggtgcaggcg 1980
          ctgttgccgg tgctgtgcca ggccacggc ttgaccccctc agcaggtggt ggccatcgcc 2040
          agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
          ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
          cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
          aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
          gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
          aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
          aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
          gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
          aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
          gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
          aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
          tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
          gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
           <![CDATA[ <210> 247]]>
           <![CDATA[ <211> 2829]]>
           <![CDATA[ <212>DNA]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> PD-1-right TALEN]]>
           <![CDATA[ <400> 247]]>
          atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
          gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
          cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
          ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
          ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
          gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
          acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
          attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
          acgggtgccc cgctcaactt gacccccgag caagtcgtcg caatcgccag ccatgatgga 540
          gggaagcaag ccctcgaaac cgtgcagcgg ttgcttcctg tgctctgcca ggcccacggc 600
          cttacccctc agcaggtggt ggccatcgca agtaacggag gaggaaagca agccttggag 660
          acagtgcagc gcctgttgcc cgtgctgtgc caggcacacg gcctcacacc agagcaggtc 720
          gtggccattg cctcccatga cggggggaaa caggctctgg agaccgtcca gaggctgctg 780
          cccgtcctct gtcaagctca cggcctgact ccccaacaag tggtcgccat cgcctctaat 840
          ggcggcggga agcaggcact ggaaacagtg cagagactgc tccctgtgct ttgccaagct 900
          catgggttga ccccccaaca ggtcgtcgct attgcctcaa acgggggggg caagcaggcc 960
          cttgagactg tgcagaggct gttgccagtg ctgtgtcagg ctcacgggct cactccacaa 1020
          caggtggtcg caattgccag caacggcggc ggaaagcaag ctcttgaaac cgtgcaacgc 1080
          ctcctgcccg tgctctgtca ggctcatggc ctgacaccac aacaagtcgt ggccatcgcc 1140
          agtaataatg gcgggaaaca ggctcttgag accgtccaga ggctgctccc agtgctctgc 1200
          caggcaacg ggctgacccc cgagcaggtg gtggctatcg ccagcaatat tggggggcaag 1260
          caggccctgg aaacagtcca ggccctgctg ccagtgcttt gccaggctca cgggctcact 1320
          ccccagcagg tcgtggcaat cgcctccaac ggcggaggga agcaggctct ggagaccgtg 1380
          cagagactgc tgcccgtctt gtgccaggcc cacggactca cacctgaaca ggtcgtcgcc 1440
          attgcctctc acgatggggg caaacaagcc ctggagacag tgcagcggct gttgcctgtg 1500
          ttgtgccaag cccacggctt gactcctcaa caagtggtcg ccatcgcctc aaatggcggc 1560
          ggaaaacaag ctctggagac agtgcagagg ttgctgcccg tcctctgcca agcccacggc 1620
          ctgactcccc aacaggtcgt cgccattgcc agcaacaacg gaggaaagca ggctctcgaa 1680
          actgtgcagc ggctgcttcc tgtgctgtgt caggctcatg ggctgacccc cgagcaagtg 1740
          gtggctattg cctctaatgg aggcaagcaa gcccttgaga cagtccagag gctgttgcca 1800
          gtgctgtgcc aggcccacgg gctcacaccc cagcaggtgg tcgccatcgc cagtaacaac 1860
          gggggcaaac aggcattgga aaccgtccag cgcctgcttc cagtgctctg ccaggcacac 1920
          ggactgacac ccgaacaggt ggtggccatt gcatcccatg atgggggcaa gcaggccctg 1980
          gagaccgtgc agagactcct gccagtgttg tgccaagctc acggcctcac ccctcagcaa 2040
          gtcgtggcca tcgcctcaaa cggggggggc cggcctgcac tggagagcat tgttgcccag 2100
          ttatctcgcc ctgatccggc gttggccgcg ttgaccaacg accacctcgt cgccttggcc 2160
          tgcctcggcg ggcgtcctgc gctggatgca gtgaaaaagg gattggggga tcctatcagc 2220
          cgttcccagc tggtgaagtc cgagctggag gagaagaaat ccgagttgag gcacaagctg 2280
          aagtacgtgc cccacgagta catcgagctg atcgagatcg cccggaacag cacccaggac 2340
          cgtatcctgg agatgaaggt gatggagttc ttcatgaagg tgtacggcta caggggcaag 2400
          cacctgggcg gctccaggaa gcccgacggc gccatctaca ccgtgggctc ccccatcgac 2460
          tacggcgtga tcgtggacac caaggcctac tccggcggct acaacctgcc catcggccag 2520
          gccgacgaaa tgcagaggta cgtggaggag aaccagacca ggaacaagca catcaaccccc 2580
          aacgagtggt ggaaggtgta cccctccagc gtgaccgagt tcaagttcct gttcgtgtcc 2640
          ggccacttca agggcaacta caaggcccag ctgaccaggc tgaaccacat caccaactgc 2700
          aacggcgccg tgctgtccgt ggaggagctc ctgatcggcg gcgagatgat caaggccggc 2760
          accctgaccc tggaggaggt gaggaggaag ttcaacaacg gcgagatcaa cttcgcggcc 2820
          gactgataa 2829
          
      

Claims (135)

A method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: processing a tumor sample into tumor fragments or processing a tumor sample obtained from the individual into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual or patient; (b) from (a ) for the first TIL population to select CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL to obtain enriched CD39/CD69 double-negative TIL population; (c) as the case may be, enrich the CD39/CD69 double The negative TIL population is added to the closed system; (d) performing a first expansion by culturing the CD39/CD69 double negative TIL population enriched in IL-2 containing cell culture medium to generate a second TIL population, Wherein the first amplification is carried out in a closed container providing a first gas-permeable surface area, wherein the first amplification is carried out for about 3-14 days to obtain the second TIL population, and wherein from step (c) to step ( The conversion of d) is carried out optionally without opening the system; (e) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen presenting cells (APCs) A second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) collected from step ( e) the third TIL population obtained, wherein the transition from step (e) to step (f) is optionally carried out without opening the system; (g) the collected TIL from step (f) The third TIL population is transferred to an infusion bag, wherein transfer from step (f) to (g) is optionally performed without opening the system; (h) cryopreservation of the TIL from step (g) using a cryopreservation process an infusion bag comprising the collected third population of TILs; (i) administering to the individual a therapeutically effective dose of the third population of TILs from the infusion bag in step (h); and (j) optionally during the administration At any time prior to step (i), genetically modifying the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, the second TIL population and/or the third TIL population such that the administered The third TIL population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: processing a tumor sample into tumor fragments or processing a tumor sample obtained from the individual into a tumor digest to obtain and/or receive a first population of TILs derived from a tumor resected from the individual or patient; (b) optionally The tumor fragments or tumor digests are added to the closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, Optionally wherein the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine (Perifosine), Oridonin, Herbacetin, Tehranolide, Isoliquiritigenin, Scutellarin and Honokiol, and generating a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is optionally performed in a closed vessel providing a first gas permeable surface area, wherein The first amplification is performed for about 3-14 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally performed without opening the system; (d) by A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 - 14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein from step ( c) the transition to step (d) is optionally carried out without opening the system; (e) collecting the third TIL population obtained from step (e), wherein from step (d) to step (e) (f) transferring the collected third TIL population from step (f) to an infusion bag, wherein the transfer from step (e) to (f) (g) cryopreserving the infusion bag comprising the collected third TIL population from step (f) using a cryopreservation process; (h) administering treatment to the individual The effective dose comes from step (g) the third TIL population of the infusion bag; and (i) optionally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time prior to the administering step (h), Such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: processing a tumor sample into tumor fragments or processing a tumor sample obtained from the individual into a tumor digest to obtain and/or receive a first population of TILs derived from a tumor resected from the individual or patient; (b) optionally The tumor fragments or tumor digests are added to the closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 to generate a second TIL population, wherein the The first amplification system is optionally carried out in a closed container providing a first gas-permeable surface area, wherein the first amplification is carried out for about 3-14 days to obtain the second TIL population, and wherein from step (b) to step ( The conversion of c) is carried out optionally without opening the system; (d) by supplementing the system with additional IL-2, OKT-3, antigen presenting cell (APC) and protein kinase B (AKT) inhibitors The cell culture medium of the second TIL population is used for the second expansion, wherein the AKT inhibitor is selected from the group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, nosytin, tenoride, isoliquiritigenin, luteinin, and honokiol to generate a third TIL population, which is enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population, wherein the second expansion is carried out for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein The second amplification is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e ) collecting the third population of TILs obtained from step (e), wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) will be obtained from step (f) The collected third TIL population is transferred to an infusion bag, wherein the transfer from steps (e) to (f) is optionally performed without opening the system; (g) cryopreservation from step (g) using a cryopreservation process (f) the infusion bag comprising the collected third TIL population; (h) administering to the subject a therapeutically effective dose of the third TIL population from the infusion bag in step (g); and (i) depending on Situation genetically modify the first TIL population, the second TIL population and/or the third TIL population at any time prior to the administering step (h), such that the administered third TIL population comprises genetically modified TIL, the genes Modified TILs include genetic modifications that reduce the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: processing a tumor sample into tumor fragments or processing a tumor sample obtained from the individual into a tumor digest to obtain and/or receive a first population of TILs derived from a tumor resected from the individual or patient; (b) optionally The tumor fragments or tumor digests are added to the closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, Optionally, wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, dongling Grassyrin, Gossytin, Tenolid, Isoliquiritigenin, Phetatin, and Honokiol to generate a second population of TILs enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative A population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, and wherein from step ( b) The transition to step (c) is carried out optionally without opening the system; (d) by adding additional IL-2, OKT-3, antigen presenting cells (APC) and protein kinase B (AKT ) inhibitor to supplement the cell culture medium of the second TIL population for the second expansion, optionally wherein the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068 , AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenolid, isoliquiritigenin, luteinin and honokiol to generate the third TIL population , which is a TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative, wherein the second expansion is carried out for about 7-14 days to obtain the third TIL population, wherein the third TIL population is therapeutic A population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally performed without opening the system (e) collecting the third TIL population obtained from step (e), wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) The collected third TIL population from step (f) is transferred to infusion bag, wherein the transfer from step (e) to (f) is optionally carried out without opening the system; (g) cryopreservation from step (f) comprising the collected third TIL is cryopreserved using a cryopreservation process (h) administering to the individual a therapeutically effective dose of a third population of TILs from the infusion bag in step (g); and (i) optionally any TIL prior to the administering step (h). Temporally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population such that the administered third TIL population comprises genetically modified TILs comprising genetically modified TILs comprising reduced CD39 and genetic modification of CD69 expression. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) by processing a tumor sample obtained from the individual into multiple tumor fragments or processing a tumor sample obtained from an individual into a tumor digest to obtain a first population of TILs derived from a tumor resected from an individual; (b) selecting CD39 ^LO / CD69 ^LO and/or CD39/CD69 double-negative TIL to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population; (c) enrich CD39 ^LO /CD69 ^LO and/or The CD39/CD69 double negative TIL population is added to the closed system; (d) by culturing the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population enriched in IL-2 containing cell culture medium A first amplification to produce a second population of TILs, wherein the first amplification is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain the second TIL Two TIL populations, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) by presenting cells with additional IL-2, OKT-3 and antigen (APC) supplementing the cell culture medium of the second TIL population for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the second Amplification is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) collected from The third TIL population obtained in step (e), wherein the transition from step (e) to step (f) is optionally performed without opening the system; The collected third TIL population is transferred to an infusion bag, wherein the transfer from step (f) to (g) is optionally performed without opening the system; (h) cryopreservation from step (g) using a cryopreservation process the infusion bag comprising the collected TIL population; (i) administering to the individual a therapeutically effective dose of the third TIL population from the infusion bag in step (h); and (j) optionally at the administration At any time prior to step (i), genetically modifying the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, the second TIL population and/or the third TIL population such that the administered The third TIL population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof, comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) by processing a tumor sample obtained from the individual or processing a tumor sample obtained from the individual into tumor digests to obtain a first TIL population derived from a tumor resected from the individual; (b) adding the tumor fragments or tumor digests, as appropriate into a closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected Free group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, resinthin, Tenolide, isoliquiritigenin, lutein and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplified is optionally performed in a closed vessel providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) (d) a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen presenting cells (APCs) , to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally carried out without opening the system; (e) collecting the third TIL population obtained from step (e), wherein from step (d) The transition to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (f) to an infusion bag, wherein from step (e) to The transfer of (f) is performed without opening the system, as appropriate; (g) cryopreservation of the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h) transfer to the individual administering a therapeutically effective dose of the third TIL population from the infusion bag in step (g); and (i) optionally genetically modifying the first TIL population, the second TIL population at any time prior to the administering step (h); The second TIL population and/or the third TIL population such that the administered third TIL population comprises genetically modified TILs comprising genetic modifications that reduce expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof, comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) by processing a tumor sample obtained from the individual or processing a tumor sample obtained from the individual into tumor digests to obtain a first TIL population derived from a tumor resected from the individual; (b) adding the tumor fragments or tumor digests, as appropriate to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is optionally provided Performed in a closed container of the first air-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally performed at different times (d) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors Second amplification, optionally wherein the AKT inhibitor is selected from the group consisting of: paltaxetide, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perid Fuxin, Rubescensin A, Gossin, Tenoride, Isoliquiritigenin, Parcetrin and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39 /CD69 double-negative TIL population, wherein the second expansion is carried out for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally at Carried out in a closed container providing a second air-permeable surface area, and wherein the transition from step (c) to step (d) is optionally carried out without opening the system; (e) collected from step (e) The third TIL population, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) the collected third TIL population from step (f) Transfer to an infusion bag, wherein the transfer from steps (e) to (f) is carried out without opening the system as appropriate; (g) cryopreservation from step (f) containing the collected an infusion bag of a TIL population; (h) administering to the subject a therapeutically effective dose of a third TIL population from the infusion bag in step (g); and (i) optionally prior to the administering step (h) Genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time such that the administered third TIL population comprises genetically modified TILs comprising reduced CD39 and genetic modification of CD69 expression. A method for treating cancer in a patient or individual in need thereof, comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) by processing a tumor sample obtained from the individual or processing a tumor sample obtained from the individual into tumor digests to obtain a first TIL population derived from a tumor resected from the individual; (b) adding the tumor fragments or tumor digests, as appropriate into a closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected Free group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, resinthin, Tenolide, isoliquiritigenin, lutein and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplified is optionally performed in a closed vessel providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) (d) by supplementing the second TIL population with additional IL-2, OKT-3, antigen presenting cell (APC) and protein kinase B (AKT) inhibitors The cell culture medium for second expansion, where the AKT inhibitor is selected from the group consisting of pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Rubescensin, Gossin, Tenolide, Isoliquiritigenin, Parifaxin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population, wherein the second expansion is carried out for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion The augmentation is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) collected from step (e) the third TIL population obtained, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) the collected TIL from step (f) The third TIL population is transferred to the infusion bag, wherein from step (e) to ( The transfer of f) is performed optionally without opening the system; (g) cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h) administering to the individual and a therapeutically effective dose of the third TIL population from the infusion bag in step (g); and (i) optionally genetically modifying the first TIL population, the second TIL population at any time prior to the administering step (h). The TIL population and/or the third TIL population such that the administered third TIL population comprises genetically modified TILs comprising genetic modifications that reduce expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: , small biopsy, or other means used to obtain a sample containing a mixture of tumor and TIL cells from cancer in the patient or individual and/or receive a first TIL population, (b) from the first TIL population in (a) A TIL population selects CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL to obtain an enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 TIL population; (c) depending on the situation, the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations are added to the closed system; (d) by culturing the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TILs in a cell culture medium containing IL-2 population for a first amplification to produce a second TIL population, wherein the first amplification is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtaining the second TIL population, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) by using additional IL-2, OKT-3 and Antigen-presenting cells (APCs) are supplemented with the cell culture medium of the second TIL population for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein The second amplification is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f ) collecting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) is optionally performed without opening the system; (g) will be obtained from step (f) The collected third TIL population is transferred to an infusion bag, wherein the transfer from steps (e) to (f) is optionally performed without opening the system; (h) cryopreservation from step (h) using a cryopreservation process (g) the infusion bag comprising the collected TIL population; (i) administering to the subject a therapeutically effective dose of a third TIL population from the infusion bag in step (h); and (j) optionally in At any time prior to the administering step (i), genetically modifying the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, the second TIL population and/or the third TIL population such that the The third population of TILs administered comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: , small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from the cancer in the patient or individual, and/or receiving the first TIL population, Tumor digests are added to the closed system; (c) a first expansion is performed by culturing the first TIL population in cell culture medium containing IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT The inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, Gossin, tenolide, isoliquiritigenin, lutein and honokiol to generate a second TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the The first amplification system is optionally carried out in a closed container providing a first gas-permeable surface area, wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein from step (b) to step ( The conversion of c) is carried out optionally without opening the system; (d) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen presenting cells (APCs) A second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the second expansion is optionally in an enclosure providing a second gas permeable surface area (e) collecting the third TIL population obtained from step (d), wherein The transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (e) to an infusion bag, wherein The transfer of (e) to (f) is performed without opening the system as appropriate; (g) cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h ) administering to the individual a therapeutically effective dose of the third TIL population from the infusion bag in step (g); and (i) optionally genetically modifying the first TIL at any time prior to the administering step (h) population, the second population of TILs and/or the third population of TILs such that the administered third population of TILs comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69 . A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: , small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from the cancer in the patient or individual, and/or receiving the first TIL population, adding tumor digests to the closed system; (c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is optionally in a closed vessel providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is (d) by supplementing the second TIL population with additional IL-2, OKT-3, antigen presenting cell (APC) and protein kinase B (AKT) inhibitors Cell culture medium is used for the second expansion, where the AKT inhibitor is selected from the group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenoride, isoliquiritigenin, luteinin and honokiol to generate a third TIL population which is enriched for CD39 ^LO /CD69 ^LO And/or a CD39/CD69 double-negative TIL population, wherein the second expansion is carried out for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) collected from step ( d) the third TIL population obtained, wherein the transition from step (d) to step (e) is optionally carried out without opening the system; (f) the collected TIL from step (e) The third TIL population is transferred to an infusion bag, wherein the transfer from steps (e) to (f) is optionally performed without opening the system; (g) cryopreserving the TIL from step (f) using a cryopreservation process an infusion bag comprising the collected TIL population; (h) administering to the individual a therapeutically effective dose of a third TIL population from the infusion bag in step (g); and (i) optionally in the administering step ( h) at any time prior to genetically modifying the first TIL population, the second TIL population and/or the third TIL population such that the administered third TIL population comprises genetically modified TILs, the genetically modified TIL package Contains genetic modifications that reduce the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: , small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells from the cancer in the patient or individual, and/or receiving the first TIL population, Tumor digests are added to the closed system; (c) a first expansion is performed by culturing the first TIL population in cell culture medium containing IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT The inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, Gossin, tenolide, isoliquiritigenin, lutein and honokiol to generate a second TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the The first amplification system is optionally carried out in a closed container providing a first gas-permeable surface area, wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein from step (b) to step ( The conversion of c) is carried out optionally without opening the system; (d) by supplementing the system with additional IL-2, OKT-3, antigen presenting cell (APC) and protein kinase B (AKT) inhibitors The cell culture medium of the second TIL population is used for the second expansion, wherein the AKT inhibitor is selected from the group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, nosytin, tenoride, isoliquiritigenin, luteinin, and honokiol to generate a third TIL population, which is enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population, wherein the second expansion is carried out for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein The second amplification is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e ) collecting the third population of TILs obtained from step (d), wherein the transition from step (d) to step (e) is optionally carried out without opening the system; The collected third TIL population was transferred to the infusion bag, wherein The transfer of steps (e) to (f) is optionally performed without opening the system; (g) cryopreserving the infusion bag from step (f) containing the collected TIL population using a cryopreservation process; ( h) administering to the individual a therapeutically effective dose of the third population of TILs from the infusion bag in step (g); and (i) optionally genetically modifying the first TIL population at any time prior to the administering step (h); TIL population, the second TIL population and/or the third TIL population such that the administered third TIL population comprises genetically modified TILs comprising genes that reduce the expression of CD39 and CD69 grooming. A method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) resecting a tumor from the individual or patient, The tumor comprises a first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b ) processing the tumor into a plurality of tumor fragments; (c) enzymatically digesting the plurality of tumor fragments to obtain the first TIL population; (d) selecting CD39 ^LO from the first TIL population in (c) /CD69 ^LO and/or CD39/CD69 double-negative TIL to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population; (e) enrich CD39 ^LO /CD69 ^LO and/or or a CD39/CD69 double-negative TIL population is added to the closed system; (f) by culturing the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population in a cell culture medium containing IL-2 to performing a first amplification to generate a second population of TILs, wherein the first amplification is optionally performed in a closed vessel providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain the A second population of TILs, and wherein the transition from step (e) to step (f) is optionally performed without opening the system; (g) by presenting with additional IL-2, OKT-3 and antigen cells (APC) supplemented with the cell culture medium of the second TIL population for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the first The second amplification is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) is optionally performed without opening the system; (h) collecting The third TIL population obtained from step (g), wherein the transition from step (g) to step (h) is optionally carried out without opening the system; (i) the TIL population from step (h) The collected third TIL population is transferred to an infusion bag, wherein the transfer from steps (h) to (i) is optionally performed without opening the system; (j) cryopreservation from step (i) using a cryopreservation process ) the infusion bag comprising the collected TIL population; (k) administering a therapeutically effective dose of the third TIL population from the infusion bag in step (g) to the individual or patient suffering from cancer; and (l ) optionally genetically modifying the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, the second TIL population and/or the third TIL population at any time prior to the administering step (k) The population of ILs such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) resecting a tumor from the individual or patient, The tumor comprises a first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b ) processing the tumor into a plurality of tumor fragments; (c) enzymatically digesting the plurality of tumor fragments to obtain the first TIL population; (d) optionally adding the tumor fragments or tumor digests to a closed In the system; (e) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of Constituent group: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifoxin, oridonin, cotton xanthin, tenol Lido, isoliquiritigenin, chrysanthemum and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is based on The situation is carried out in a closed container providing a first gas-permeable surface area, wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein the transition from step (d) to step (e) depends on (f) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs), to generating a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the second expansion is optionally performed in a closed vessel providing a second gas-permeable surface area, and wherein The transition from step (e) to step (f) is optionally carried out without opening the system; (g) collecting the third TIL population obtained from step (f), wherein from step (f) to step The transformation of (g) is carried out optionally without opening the system; (h) transferring the collected third TIL population from step (g) to an infusion bag, wherein steps (g) to (h ) transfer is performed without opening the system as appropriate; (i) cryopreservation of the infusion bag containing the collected TIL population from step (h) using a cryopreservation process; (l) transfer to the patient with cancer administering a therapeutically effective dose of the third TIL population from the infusion bag in step (h) to the individual or patient; and (k) optionally genetically modifying the first TIL at any time prior to the administering step (l) group, the second TI The L population and/or the third TIL population such that the administered third TIL population comprises genetically modified TILs comprising genetic modifications that reduce expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) resecting a tumor from the individual or patient, The tumor comprises a first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b ) processing the tumor into a plurality of tumor fragments; (c) enzymatically digesting the plurality of tumor fragments to obtain the first TIL population; (d) optionally adding the tumor fragments or tumor digests to a closed In the system; (e) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion optionally provides the first Carried out in a closed container with a gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (e) to step (f) is optionally performed without opening the (f) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors to perform a second TIL population amplified, optionally wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine , Rubescensin A, Gossin, Tenolide, Isoliquiritigenin, Parcetrin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 A double negative TIL population, wherein the second expansion is carried out for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally provided in the second TIL population (2) carried out in a closed container with a gas-permeable surface area, and wherein the transition from step (e) to step (f) is carried out optionally without opening the system; Three TIL populations, wherein the transition from step (f) to step (g) is optionally carried out without opening the system; (h) transferring the collected third TIL population from step (g) to an infusion bag, wherein the transfer from steps (g) to (h) is optionally performed without opening the system; (i) cryopreserving the population comprising the collected TILs from step (h) using a cryopreservation process (j) administering a therapeutically effective dose of the third TIL population from the infusion bag in step (h) to the individual or patient with cancer; and (k) optionally in the administering step ( l) before any Temporally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population such that the administered third TIL population comprises genetically modified TILs comprising genetically modified TILs comprising reduced CD39 and genetic modification of CD69 expression. A method for treating cancer in a patient or individual in need thereof comprising administering a modified population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) resecting a tumor from the individual or patient, The tumor comprises a first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b ) processing the tumor into a plurality of tumor fragments; (c) enzymatically digesting the plurality of tumor fragments to obtain the first TIL population; (d) optionally adding the tumor fragments or tumor digests to a closed In the system; (e) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of Constituent group: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, tenol Lido, isoliquiritigenin, chrysanthemum and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is based on The situation is carried out in a closed container providing a first gas-permeable surface area, wherein the first amplification is carried out for about 3-14 days to obtain the second TIL population, and wherein the transition from step (d) to step (e) depends on (f) by supplementing cells of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors Medium for the second amplification, where the AKT inhibitor is optionally selected from the group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976 , Perifosine, Rubescensin, Gossin, Tenolide, Isoliquiritigenin, Radixin and Honokiol to generate a third TIL population, which is enriched for CD39 ^LO /CD69 ^LO and /or CD39/CD69 double-negative TIL population, wherein the second expansion is carried out for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally in a closed container providing a second gas-permeable surface area, and wherein the transition from step (e) to step (f) is optionally performed without opening the system; (g) collected from step (f) ) obtained the third TIL population, wherein from step ( f) the transition to step (g) is carried out optionally without opening the system; (h) transferring the collected third population of TILs from step (g) to an infusion bag, wherein from step (g) ) to (h) is transferred as appropriate without opening the system; (i) cryopreserving the infusion bag containing the collected TIL population from step (h) using a cryopreservation process; (j) transferring to administering a therapeutically effective dose of the third population of TILs from the infusion bag in step (h) to the individual or patient with cancer; and (k) optionally genetically modified at any time prior to the administering step (l) The first population of TILs, the second population of TILs and/or the third population of TILs, such that the administered third population of TILs comprises genetically modified TILs comprising reduced CD39 and CD69 Genetic modification of expression. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (b) from the first TIL population in (a) Selecting CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations; (c) making the enriched CD39 ^LO /CD69 ^LO and and/or the CD39/CD69 double negative TIL population is contacted with a first cell culture medium; (d) initial expansion of the CD39/CD69 ^{LO and} /or CD39/CD69 double negative TIL population enriched in the first cell culture medium increase (or initiate a first expansion) to obtain a second population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells ( APC), wherein the initial first expansion is performed for a period of 1 to 8 days; (e) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population; wherein the The second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and APC; and wherein the rapid expansion is performed for a period of 14 days or less, optionally the rapid second expansion can be performed during the rapid second expansion 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of multiplication; (f) collecting the third TIL population; (g) adding to the administering a therapeutically effective portion of the third TIL population to an individual or patient with cancer; and (h) optionally genetically modifying the enriched CD39 ^LO /CD69 ^LO and/or at any time prior to the administering step (g) CD39/CD69 double negative TIL population, the second TIL population and/or the third TIL population, such that the administered third TIL population comprises genetically modified TILs comprising a CD39-lowering TIL and genetic modification of CD69 expression. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: , small biopsy or other means for obtaining a sample from the individual or patient containing a mixture of tumor and TIL cells to obtain and/or receive the first TIL population; (b) by including IL-2, optionally The first TIL population was cultured in a cell culture medium of OKT-3 (anti-CD3 antibody), optionally antigen-presenting cells (APC) and protein kinase B (AKT) inhibitor for initial expansion (or initiation of the first TIL population). Amplification), where the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifol New, oridonin, gossin, tenoride, isoliquiritigenin, chrysanthemum, and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/ CD69 double negative TIL population, wherein the first expansion is optionally performed in a closed container providing a first gas permeable surface area, wherein the initial first expansion is performed for about 1-8 days to obtain the second TIL population , and wherein the transition from step (a) to step (b) is optionally performed without opening the system; (c) performing a rapid second expansion of the second TIL population in a second cell culture medium, obtaining a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and APCs; and wherein the rapid expansion is performed for a period of 14 days or less, optionally the rapid second Amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second amplification; (d) collecting the first three TIL populations; (e) administering a therapeutically effective portion of the third TIL population to the individual or patient with cancer; and (f) optionally genetically modifying the third TIL population at any time prior to the administering step (e). A population of TILs, the second population of TILs, and/or the third population of TILs, such that the administered third population of TILs comprises genetically modified TILs comprising reduced expression of CD39 and CD69 genetic modification. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: Obtaining and/or receiving a first TIL population by means of obtaining a sample containing a mixture of tumor and TIL cells from the individual or patient, a small biopsy, or other means; (b) performing the first TIL in a first cell culture medium Initial expansion of a population (or initiation of a first expansion) to obtain a second population of TILs, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally an antigen presenting cells (APCs), wherein the initial first expansion is performed for a period of 1 to 8 days; (c) performing a rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium Comprising IL-2, OKT-3 (anti-CD3 antibody), APC and protein kinase B (AKT) inhibitors, optionally wherein the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183 , AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenolid, isoliquiritigenin, chrysanthemum and honokiol, To generate a third population of TILs that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion Amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second amplification; (d) collecting the third (e) administering a therapeutically effective portion of the third TIL population to the individual or patient with cancer; and (f) optionally genetically modifying the first TIL population at any time prior to the administering step (e). TIL population, the second TIL population and/or the third TIL population such that the administered third TIL population comprises genetically modified TILs comprising genes that reduce the expression of CD39 and CD69 grooming. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: , small biopsy or other means for obtaining a sample from the individual or patient containing a mixture of tumor and TIL cells to obtain and/or receive the first TIL population; (b) by including IL-2, optionally Initially expand (or initiate first amplification), optionally wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, piperase Lifusin, Rubescensin, Gossin, Tenolide, Isoliquiritigenin, Paretin, and Honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first expansion is optionally performed in a closed container providing a first gas permeable surface area, wherein the initial first expansion is performed for about 1-8 days to obtain the second A population of TILs, and wherein the transition from step (a) to step (b) is optionally performed without opening the system; (c) performing a rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), APC and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: Seti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin , lutein, and honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the rapid expansion is performed for a period of about 14 days or less , optionally the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second amplification (d) collecting the third TIL population; (e) administering a therapeutically effective portion of the third TIL population to the individual or patient with cancer; and (f) optionally prior to the administering step (e) genetically modifying the first TIL population, the second TIL population and/or the third TIL population at any time , such that the administered third TIL population comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) resecting a tumor from the cancer in the individual or patient, The tumor comprises a first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b ) fragmenting the tumor into tumor fragments or processing the tumor into tumor digests; (c) selecting CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs from the first TIL population of the tumor fragments to obtain Enriching CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations; (c) contacting the tumor fragments with a first cell culture medium; (d) performing the enrichment for CD39 ^LO in the first cell culture medium / Initial expansion of CD69 ^LO and/or CD39/CD69 double negative TIL population (or initiation of first expansion) to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody) and optionally antigen presenting cells (APC), wherein the initial first expansion is performed for a period of 1 to 8 days; (e) the second TIL is performed in a second cell culture medium A rapid second expansion of the population to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and APCs; and wherein the rapid expansion is performed for 14 days or less Period of time, optionally the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second amplification (f) collecting the third TIL population; (g) administering a therapeutically effective portion of the third TIL population to the individual or patient with cancer; and (h) optionally in the administering step (g) Genetically modifying the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population, the second TIL population and/or the third TIL population at any time before such that the administered third TIL population Included are genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) resecting a tumor from the cancer in the individual or patient, The tumor comprises a first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b ) fragmenting the tumor into tumor fragments or processing the tumor into a tumor digest; (c) by combining IL-2, optionally OKT-3 (anti-CD3 antibody), optionally antigen presenting cells ( APC) and a protein kinase B (AKT) inhibitor in a first cell culture medium for initial expansion (or initiation of first expansion) by culturing the first TIL population, optionally wherein the AKT inhibitor is selected from the group consisting of The group: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, tenoli De, isoliquiritigenin, lutein and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is optional Carried out in a closed container providing a first gas-permeable surface area, wherein the initial first amplification is carried out for about 1-8 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is optionally performed without opening the system; (d) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL- 2. OKT-3 (anti-CD3 antibody) and APC; and wherein the rapid expansion is performed for a period of 14 days or less, optionally the rapid second amplification may be performed 1 day after initiation of the rapid second amplification , 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days; (e) collecting the third TIL population; (f) giving the individual or patient with cancer administering a therapeutically effective portion of the third TIL population; and (g) optionally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time prior to the administering step (f) A population such that the administered third TIL population comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) resecting a tumor from the cancer in the individual or patient, The tumor comprises a first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b ) fragmenting the tumor into tumor fragments or processing the tumor into a tumor digest; (c) performing an initial expansion (or initiating a first expansion) of the first TIL population in a first cell culture medium to obtain a second Two TIL populations, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody) and optionally antigen-presenting cells (APC), wherein the initial first expansion is performed for 1 to A period of 8 days; (d) rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), APC and protein kinase B(AKT) inhibitor, wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, piperase, as the case may be Lifusin, Rubescensin, Gossin, Tenolide, Isoliquiritigenin, Radixin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion can be performed 1 day, 2 days, 3 days after initiation of the rapid second expansion days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days; (e) collecting the third TIL population; (f) administering a therapeutically effective moiety to the individual or patient with cancer and (g) optionally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time prior to the administering step (f), such that the The third population of TILs administered comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: (a) resecting a tumor from the cancer in the individual or patient, The tumor comprises a first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample from the cancer containing a mixture of tumor and TIL cells; (b ) fragmenting the tumor into tumor fragments or processing the tumor into a tumor digest; (c) by combining IL-2, optionally OKT-3 (anti-CD3 antibody), optionally antigen presenting cells ( APC) and a protein kinase B (AKT) inhibitor in a first cell culture medium for initial expansion (or initiation of first expansion) by culturing the first TIL population, optionally wherein the AKT inhibitor is selected from the group consisting of The group: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, tenoli De, isoliquiritigenin, lutein and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is optional Carried out in a closed container providing a first gas-permeable surface area, wherein the initial first amplification is carried out for about 1-8 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is (d) performing a rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti- CD3 antibody), APC and protein kinase B (AKT) inhibitors, where the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930 as appropriate , MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenoride, isoliquiritigenin, luteinin and honokiol to produce the third TIL population, which is rich in Collecting CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion can be performed after the rapid second expansion 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start; (e) collecting the third TIL population; A therapeutically effective portion of the third TIL population is administered to an individual or patient with cancer; and (g) as appropriate Genetically modify the first TIL population, the second TIL population, and/or the third TIL population at any time prior to the administering step (f), such that the administered third TIL population comprises genetically modified TILs , the genetically modified TILs comprise a genetic modification that reduces the expression of CD39 and CD69. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: , small biopsy, or other means used to obtain a sample containing a mixture of tumor and TIL cells from cancer in the patient or individual and/or receive a first TIL population, (b) from the first TIL population in (a) A TIL population selects CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TILs to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations; (c) by including IL-2 , OKT-3, and antigen-presenting cells (APC) in the first cell culture medium of the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population to initiate the first expansion to generate the second A TIL population, wherein the initial first expansion is performed in a container comprising a first gas-permeable surface area, wherein the initial first expansion is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (d) optionally restimulating the second TIL population with OKT-3; (e) genetically modifying the second TIL population to produce a modified second TIL population population, wherein the modified second population of TILs comprises genetic modifications that reduce the expression of CD39 and CD69, such that the second population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs; (f) by comprising The modified second TIL population is cultured in a second culture medium of IL-2, OKT-3, and APC for a rapid second expansion to produce a third TIL population, wherein the rapid second expansion is performed for about 14 days or A shorter second period of time to obtain a therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population comprising genetic modifications that reduce the expression of CD39 and CD69 such that the third population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs; (g) collecting the third population of TILs; and (h) administering a therapeutically effective portion of the third population of TILs to the individual or patient with cancer. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: , small biopsy or other means for obtaining a sample containing a mixture of tumor and TIL cells from the cancer in the patient or individual and/or receiving a first TIL population, (b) by including IL-2, The initial first expansion is performed by culturing the first TIL population in a first cell culture medium of OKT-3, antigen presenting cells (APCs) and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from The group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, Nolide, isoliquiritigenin, lutein and honokiol to produce a second TIL population enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplified The expansion is performed in a vessel comprising a first gas-permeable surface area, wherein the initial first expansion is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the (c) optionally restimulating the second TIL population with OKT-3; (d) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population The population comprises genetic modifications that reduce the expression of CD39 and CD69 such that the second population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs; (e) by including IL-2, OKT-3 and APC The modified second TIL population is cultured in a second culture medium for rapid second expansion to produce a third TIL population, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population comprising genetic modifications that reduce the expression of CD39 and CD69, such that the third population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL; (f) collecting the third population of TILs; and (g) administering to the individual or patient with cancer a therapeutically effective portion of the third population of TILs. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: , small biopsy or other means for obtaining a sample containing a mixture of tumor and TIL cells from the cancer in the patient or individual and/or receiving a first TIL population, (b) by including IL-2, The first TIL population is cultured in a second cell culture medium of OKT-3 and antigen-presenting cells (APCs) to perform an initial first expansion to produce a second TIL population, wherein the initial first expansion is in a culture medium comprising the second TIL population. carried out in a container with a gas-permeable surface area, wherein the initial first expansion is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) optionally restimulating the second TIL population with OKT-3; (d) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises reduced CD39 and Genetic modification of CD69 expression such that the second population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs; (e) by including IL-2, OKT-3, APC and protein kinase B (AKT ) inhibitor in a second cell culture medium for rapid second expansion by culturing the modified second TIL population, optionally wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenoride, isoliquiritigenin, chrysanthemum and honokiol , to generate a third TIL population that is CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population enriched, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain treatment Sexual TIL population, wherein the third TIL population is a therapeutic TIL population comprising genetic modifications that reduce the expression of CD39 and CD69, such that the third population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL; (f ) collecting the third population of TILs; and (g) administering a therapeutically effective portion of the third population of TILs to the individual or patient with cancer. A method for treating cancer in a patient or individual in need thereof comprising administering a population of tumor infiltrating lymphocytes (TIL), the method comprising the steps of: , small biopsy or other means for obtaining a sample containing a mixture of tumor and TIL cells from the cancer in the patient or individual and/or receiving a first TIL population, (b) by including IL-2, The initial first expansion is performed by culturing the first TIL population in a first cell culture medium of OKT-3, antigen presenting cells (APCs) and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from The group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, Nolide, isoliquiritigenin, lutein and honokiol to produce a second TIL population enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplified The expansion is performed in a vessel comprising a first gas-permeable surface area, wherein the initial first expansion is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the (c) optionally restimulating the second TIL population with OKT-3; (d) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population The population comprises genetic modifications that reduce the expression of CD39 and CD69 such that the second population comprises CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs; (e) by including IL-2, OKT-3, APC and Rapid second expansion is performed by culturing the modified second TIL population in a second cell culture medium of a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: pataxerti , GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin, Huangpi and honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the rapid second expansion is performed for about 14 days or less. Two periods of time to obtain a therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population comprising genetic modifications that reduce the expression of CD39 and CD69, such that the third population comprises C D39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TILs; (f) collecting the third TIL population; (g) administering a therapeutically effective portion of the third TIL population to the individual or patient suffering from cancer. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or dividing a tumor obtained from an individual into processing the sample into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual's cancer; (b) selecting CD39 ^LO /CD69 ^LO and /or CD39/CD69 double-negative TIL to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population; (c) by including IL-2, OKT-3 and antigen-presenting cells (APC ) in the first cell culture medium of the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population to perform the initial first expansion to generate the second TIL population, wherein the initial first expansion The expansion is carried out in a container comprising a first gas-permeable surface area, wherein the initial first expansion is performed for a first period of about 1 to 7/8 days to obtain the second TIL population, wherein the number of the second TIL population larger than the first TIL population; (d) performing a rapid second expansion by culturing the second TIL population in a second medium comprising IL-2, OKT-3, and APCs to produce a third TIL population, wherein The number of APCs added in the rapid second amplification is at least twice the number of APCs added in step (b), wherein the rapid second amplification is performed for a second period of about 1 to 11 days to obtain the a third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container comprising a second gas-permeable surface area; (e) collecting the treatment obtained from step (d) (f) transferring the collected TIL population from step (e) to an infusion bag, and (g) optionally genetically modifying the enriched CD39 ^LO / CD69 ^LO and/or CD39/CD69 double negative TIL population and/or the second TIL population and/or the third TIL population, such that the therapeutic TIL population comprises genetically modified TILs, the genetically modified TILs Genetic modifications that reduce the expression of CD39 and CD69 are included. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or dividing a tumor obtained from an individual into The sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual's cancer; Initiating the first expansion by culturing the first TIL population in a first cell culture medium of a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693 , GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin A, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Magnolol, to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the initial first amplification is in a container comprising a first gas permeable surface area wherein the initial first amplification is performed for a first period of about 1 to 7/8 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) by A rapid second expansion is performed by culturing the second TIL population in a second medium comprising IL-2, OKT-3, and APCs to produce a third TIL population, wherein the APCs added in the rapid second expansion The number is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain a third TIL population, wherein the third TIL population is A therapeutic TIL population, wherein the rapid second expansion is carried out in a container comprising a second gas-permeable surface area; (d) collecting the therapeutic TIL population obtained from step (d); (e) converting the population from step (e) The collected TIL population is transferred to an infusion bag; and (f) optionally genetically modifying the first TIL population, the second TIL population, and/or the third TIL population at any time prior to the collecting step (e) , such that the therapeutic TIL population comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or dividing a tumor obtained from an individual into The sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual's cancer; The initial first expansion is performed by culturing the first TIL population in a second cell culture medium to produce a second TIL population, wherein the initial first expansion is performed in a vessel comprising a first gas permeable surface area, wherein the initial first expansion An expansion is carried out for a first period of about 1 to 7/8 days to obtain the second TIL population, wherein the number of the second TIL population is greater than the first TIL population; (c) by including IL-2, OKT -3. Rapid second expansion by culturing the second TIL population in a second medium of APC and protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: patase For, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin, Yellow acetin and honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the number of APCs added in this rapid second expansion is At least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the therapeutic TIL population, wherein the third TIL population is therapeutic TIL population, wherein the rapid second expansion is performed in a container comprising a second gas-permeable surface area; (d) collecting the therapeutic TIL population obtained from step (d); (e) converting the TIL population from step (e); transferring the collected TIL population to an infusion bag; and (f) optionally genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time prior to the collecting step (e) A population such that the therapeutic TIL population comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or dividing a tumor obtained from an individual into The sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual's cancer; Initiating the first expansion by culturing the first TIL population in a first cell culture medium of a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693 , GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin A, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Magnolol, to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the initial first amplification is in a container comprising a first gas permeable surface area wherein the initial first amplification is performed for a first period of about 1 to 7/8 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) by A rapid second expansion is performed by culturing the second TIL population in a second medium comprising IL-2, OKT-3, APC, and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from The group consisting of: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, Nolide, isoliquiritigenin, lutein and honokiol to generate a third TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein in the rapid second expansion The number of APCs added is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the therapeutic TIL population, wherein The third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container comprising a second gas-permeable surface area; (d) collecting the therapeutic TIL population obtained from step (d); (e ) transferring the collected TIL population from step (e) to an infusion bag; and (f) optionally genetically modifying the first TIL population and/or the second TIL population at any time prior to the collecting step (e) population and/or the third TIL population such that the therapeutic T The IL population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population comprising the steps of: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by The obtained tumor sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from an individual's or patient's cancer; (b) selecting CD39 ^LO / CD69 ^LO and/or CD39/CD69 double-negative TILs to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations; (c) the CD39 ^LO /CD69 ^LO and/or CD39/ The CD69 double negative TIL population is added to the closed system; (d) a first expansion is performed by culturing the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population enriched in IL-2 containing cell culture medium to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain a second population of TILs, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) by supplementing with additional IL-2, OKT-3 and antigen presenting cells (APCs) The cell culture medium of the second TIL population is used to perform a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, wherein the third TIL population is therapeutic A population of TILs, wherein the second expansion is optionally performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) is optionally performed without opening the system (f) collecting the third TIL population obtained from step (e), wherein the transition from step (e) to step (f) is optionally performed without opening the system; (g) transfer of the collected third population of TILs from step (f) to an infusion bag, wherein the transfer from steps (f) to (g) is optionally performed without opening the system; and (h) optionally At any time before the collection step (f), genetically modify the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population and/or the second TIL population and/or the third TIL population, such that The third TIL population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population comprising the steps of: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by The tumor samples obtained are processed into tumor digests to obtain and/or receive a first population of TILs derived from tumors resected from an individual's or patient's cancer; (b) adding these tumor fragments or tumor digests to the closed system as appropriate wherein; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of The group: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, tenoli De, isoliquiritigenin, lutein and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is optional In a closed container providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-14 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optional Performed without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate A third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally provided in the second carried out in a closed container with a gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally carried out without opening the system; (e) collecting the third TIL population, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (e) to infusion bag, wherein the transfer from steps (e) to (f) is optionally performed without opening the system; and (g) optionally genetically modifying the first TIL at any time prior to the collecting step (f) population and/or the second population of TILs and/or the third population of TILs such that the third population of TILs comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population comprising the steps of: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by The tumor samples obtained are processed into tumor digests to obtain and/or receive a first population of TILs derived from tumors resected from an individual's or patient's cancer; (b) adding these tumor fragments or tumor digests to the closed system as appropriate wherein; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is optionally provided with a first gas permeable In a closed container of surface area, wherein the first amplification is carried out for about 3-14 days to obtain the second TIL population, and wherein the transition from step (c) to step (d) is optionally performed without opening the system (d) a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors , wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, Rubescensin, Gossin, Tenolide, Isoliquiritigenin, Radixin, and Honokiol to generate a third TIL population that is CD39 ^LO /CD69 ^LO and/or CD39/CD69 double Negative TIL population, wherein the second expansion is carried out for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally provided in the second carried out in a closed container with a gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally carried out without opening the system; (e) collecting the third TIL population, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (e) to infusion bag, wherein the transfer from steps (e) to (f) is optionally performed without opening the system; and (g) optionally genetically modifying the first TIL at any time prior to the collecting step (f) population and/or the second population of TILs and/or the third population of TILs such that the third population of TILs comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population comprising the steps of: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by The tumor samples obtained are processed into tumor digests to obtain and/or receive a first population of TILs derived from tumors resected from an individual's or patient's cancer; (b) adding these tumor fragments or tumor digests to the closed system as appropriate wherein; (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of The group: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, tenoli De, isoliquiritigenin, lutein and honokiol to generate a second TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification is optional In a closed container providing a first gas-permeable surface area, wherein the first amplification is performed for about 3-14 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optional Carried out without opening the system; (d) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors to perform a second amplification, optionally wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Rubescensin, Gossytin, Tenolide, Isoliquiritigenin, Radixin, and Honokiol to generate a third TIL population enriched for CD39 ^LO /CD69 ^LO and/ or a CD39/CD69 double-negative TIL population, wherein the second expansion is carried out for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is based on The case is carried out in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is carried out optionally without opening the system; (e) collected from step (d) The third TIL population obtained, wherein the transition from step (d) to step (e) is optionally carried out without opening the system; (f) the collected third TIL from step (e) The TIL population is transferred to the infusion bag, wherein from steps (e) to (f) transferring is optionally performed without opening the system; and (g) optionally genetically modifying the first TIL population and/or the second TIL population and/or the A third population of TILs such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population comprising the steps of: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or by The obtained tumor sample is processed into a tumor digest to obtain a first TIL population derived from a tumor resected from the individual's cancer; b) selecting CD39 ^LO /CD69 ^LO and/or CD39 from the first TIL population in (a) /CD69 double negative TILs to obtain enriched (i) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations; (c) enrich CD39 ^LO /CD69 ^LO and/or CD39/CD69 The double negative TIL population is added to the closed system; (d) a first expansion is performed by culturing the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population enriched in IL-2 containing cell culture medium to produce a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain a second population of TILs, and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) by supplementing with additional IL-2, OKT-3 and antigen presenting cells (APCs) The cell culture medium of the second TIL population is used to perform a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the second expansion depends on The case is carried out in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) is carried out optionally without opening the system; (f) collected from step (e) The third TIL population obtained, wherein the transition from step (e) to step (f) is optionally carried out without opening the system; (g) the collected third TIL from step (f) The TIL population is transferred to an infusion bag, wherein the transfer from steps (f) to (g) is optionally performed without opening the system; and (h) optionally at any time prior to the collecting step (f) modifying the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population and/or the second TIL population and/or the third TIL population such that the third TIL population comprises genetically modified TILs, The genetically modified TILs comprise genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) by processing a tumor sample obtained from the individual into a plurality of tumor fragments or by processing a tumor sample obtained from the individual into a tumor digest to obtain a first TIL population derived from a tumor resected from the individual; (b) adding the tumor fragments or tumor digest, as appropriate, to the closed system; (c) The first expansion is performed by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: Seti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin , yellow acetin and honokiol, to produce a second TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification system is optionally provided in the first gas permeable In a closed container of surface area, wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally performed without opening the system (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen-presenting cells (APCs) to produce a third TIL population, Wherein the second amplification is carried out for about 7-11 days to obtain the third TIL population, wherein the second amplification is optionally performed in a closed container providing a second gas-permeable surface area, and wherein steps (c) to The conversion of step (d) is optionally carried out without opening the system; (e) collecting the third TIL population obtained from step (e), wherein the conversion from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (f) to an infusion bag, wherein the transfer from steps (e) to (f) is optional (g) cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h) administering to the individual a therapeutically effective dose of the TIL from step (g) the third TIL population of the infusion bag; and (i) optionally genetically modifying the first TIL population and/or the second TIL population and/or at any time prior to the administering step (h) The third population of TILs is such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) by processing a tumor sample obtained from the individual into a plurality of tumor fragments or by processing a tumor sample obtained from the individual into a tumor digest to obtain a first TIL population derived from a tumor resected from the individual; (b) adding the tumor fragments or tumor digest, as appropriate, to the closed system; (c) A first expansion is performed by culturing the first TIL population in cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is optionally in a closed vessel providing a first gas-permeable surface area wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally performed without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors, optionally wherein The AKT inhibitor is selected from the group consisting of pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin , gossinthin, tenolide, isoliquiritigenin, chrysanthemum and honokiol to generate a third TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally in an enclosure providing a second gas permeable surface area (e) collecting the third TIL population obtained from step (e), wherein The transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (f) to an infusion bag, wherein The transfer of (e) to (f) is performed without opening the system as appropriate; (g) cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h ) administering to the individual a therapeutically effective dose of the third TIL population from the infusion bag in step (g); and (i) optionally genetically modifying the first TIL at any time prior to the administering step (h) population and/or the second population of TILs and/or the third population of TILs such that the administered third population of TILs comprises genetically modified TILs comprising reduced expression of CD39 and CD69 genetic modification. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) by processing a tumor sample obtained from the individual into a plurality of tumor fragments or by processing a tumor sample obtained from the individual into a tumor digest to obtain a first TIL population derived from a tumor resected from the individual; (b) adding the tumor fragments or tumor digest, as appropriate, to the closed system; (c) The first expansion is performed by culturing the first TIL population in cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: Seti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin , yellow acetin and honokiol, to produce a second TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the first amplification system is optionally provided in the first gas permeable In a closed container of surface area, wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally performed without opening the system (d) a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors , wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, Rubescensin, Gossin, Tenolide, Isoliquiritigenin, Radixin, and Honokiol to generate a third TIL population that is CD39 ^LO /CD69 ^LO and/or CD39/CD69 double Negative TIL population, wherein the second expansion is carried out for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally provided in the second in a closed container with a gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally carried out without opening the system; (e) collecting the third TIL population, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (f) to infusion bag, wherein the transfer from step (e) to (f) is at different (g) cryopreserving the infusion bag containing the collected TIL population from step (f) using a cryopreservation process; (h) administering to the individual a therapeutically effective dose of the TIL from step (g) ) of the third TIL population of the infusion bag; and (i) optionally genetically modifying the first TIL population and/or the second TIL population and/or the second TIL population at any time prior to the administering step (h) Three populations of TILs such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) by surgical excision, biopsy biopsy, core needle biopsy, mini-biopsy, or other Means of obtaining a sample containing a mixture of tumor and TIL cells from a cancer in a patient or individual Obtaining and/or receiving a first TIL population, (b) selecting CD39 ^LO /CD69 ^LO from this first TIL population in (a) and/or CD39/CD69 double-negative TILs to obtain CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations; (c) enrich CD39 ^LO /CD69 ^LO and/or CD39/ The CD69 double negative TIL population is added to the closed system; (d) the first TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative is performed by culturing the TIL population enriched in IL-2 containing cell culture medium. Expansion to produce a second population of TILs, wherein the first expansion is optionally performed in a closed vessel providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs , and wherein the transition from step (c) to step (d) is optionally performed without opening the system; (e) by adding additional IL-2, OKT-3 and antigen presenting cells (APC) Supplementing the cell culture medium of the second TIL population for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the second expansion is optionally in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) collected from step (e) ) the third TIL population obtained, wherein the transition from step (e) to step (f) is optionally carried out without opening the system; (g) the collected first TIL population from step (f) Three TIL populations are transferred to an infusion bag, wherein the transfer from steps (e) to (f) is optionally performed without opening the system; and (h) optionally at any time prior to the collecting step (f) genetically modifying the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population and/or the second TIL population and/or the third TIL population such that the third TIL population comprises genetically modified TILs , the genetically modified TILs comprise a genetic modification that reduces the expression of CD39 and CD69. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) by surgical excision, biopsy biopsy, core needle biopsy, mini-biopsy, or other means of obtaining a sample from a cancer in the patient or individual containing a mixture of tumor and TIL cells, obtaining and/or receiving a first population of TILs, (b) adding the tumor fragments or tumor digest as appropriate to the closed system (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of Group: Pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Oridonin, Gossin, Tenolide , isoliquiritigenin, chrysanthemum and honokiol to produce a second population of TILs enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplification is optionally at Provided in a closed container with a first gas-permeable surface area, wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally at (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate the second TIL population. Three TIL populations, wherein the second amplification is carried out for about 7-11 days to obtain the third TIL population, wherein the second amplification is optionally performed in a closed container providing a second gas-permeable surface area, and wherein from step (c) the transition to step (d) is optionally carried out without opening the system; (e) collecting the third TIL population obtained from step (d), wherein from step (d) to step (e ) transformation is carried out without opening the system as appropriate; (f) transferring the collected third TIL population from step (e) to an infusion bag, wherein the transferring is optionally performed without opening the system; and (g) optionally genetically modifying the first TIL population and/or the second TIL population and/or the A third population of TILs such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) by surgical excision, biopsy biopsy, core needle biopsy, mini-biopsy, or other means of obtaining a sample from a cancer in the patient or individual containing a mixture of tumor and TIL cells, obtaining and/or receiving a first population of TILs, (b) adding the tumor fragments or tumor digest as appropriate to the closed system (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion optionally provides a first gas-permeable surface In a closed container in an area, wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally performed without opening the system (d) a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors , wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, winter Isoridin, Gossytin, Tenolide, Isoliquiritigenin, Phetatin, and Honokiol to generate a third TIL population that is CD39 ^LO /CD69 ^LO enriched and/or CD39/CD69 double negative The TIL population, wherein the second expansion is carried out for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally provided after providing a second gas permeable in a closed container of the surface area, and wherein the transition from step (c) to step (d) is optionally carried out without opening the system; (e) collecting the third TIL obtained from step (d) A population, wherein the transition from step (d) to step (e) is optionally performed without opening the system; (f) transferring the collected third TIL population from step (e) to an infusion bag , wherein the transfer from steps (e) to (f) is optionally performed without opening the system; and (g) optionally genetically modifying the first TIL population at any time prior to the collecting step (e) And/or the second TIL population and/or the third TIL population, such that the third TIL population comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) by surgical excision, biopsy biopsy, core needle biopsy, mini-biopsy, or other means of obtaining a sample from a cancer in the patient or individual containing a mixture of tumor and TIL cells, obtaining and/or receiving a first population of TILs, (b) adding the tumor fragments or tumor digest as appropriate to the closed system (c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of Group: Pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Oridonin, Gossin, Tenolide , isoliquiritigenin, chrysanthemum and honokiol to produce a second population of TILs enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplification is optionally at Provided in a closed container with a first gas-permeable surface area, wherein the first amplification is carried out for about 3-11 days to obtain the second TIL population, and wherein the transition from step (b) to step (c) is optionally at (d) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors A second amplification is performed, optionally wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, piperase Lifusin, Rubescensin, Gossin, Tenolide, Isoliquiritigenin, Radixin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population, wherein the second expansion is carried out for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optional Carried out in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) is optionally done without opening the system; (e) collected from step (d) the third TIL population, wherein the transition from step (d) to step (e) is optionally carried out without opening the system; (f) the collected third TIL from step (e) The population is transferred to the infusion bag, where the transfer from steps (e) to (f) Transplanting is optionally performed without opening the system; and (g) optionally genetically modifying the first TIL population and/or the second TIL population and/or the A third population of TILs such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) resecting a tumor from a cancer in an individual or patient, the tumor comprising a first TIL population, depending on The condition is performed by surgical resection, biopsy biopsy, core biopsy, mini biopsy or other means used to obtain a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into multiple tumors fragments or tumor digests; (c) enzymatically digesting the plurality of tumor fragments to obtain the first TIL population; (d) selecting CD39 ^LO /CD69 ^LO and/or from the first TIL population in (c) or CD39/CD69 double-negative TIL to obtain enriched CD39 ^LO / CD69 ^LO and/or CD39/CD69 double-negative TIL population; (e) enrich CD39 ^LO /CD69 ^LO and/or CD39/CD69 double as appropriate The negative TIL population is added to the closed system; (f) a first expansion is performed by culturing the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population in cell culture medium containing IL-2 , to generate a second population of TILs, wherein the first expansion is optionally performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (e) to step (f) is optionally performed without opening the system; (g) by supplementing with additional IL-2, OKT-3 and antigen presenting cells (APCs) The cell culture medium of the second TIL population is used to perform a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the second expansion depends on The case is carried out in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) is carried out optionally without opening the system; (h) collected from step (g) The third TIL population obtained, wherein the transition from step (g) to step (h) is optionally carried out without opening the system; (i) the collected third TIL from step (h) transfer of the population to an infusion bag, wherein the transfer from steps (h) to (i) is optionally performed without opening the system; and (j) optionally at any time prior to the collection step (h), genetically modifying the Enriching CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations and/or the second TIL population and/or the third TIL population such that the third TIL population comprises genetically modified TILs, the Genetically modified TILs include genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) resecting a tumor from a cancer in an individual or patient, the tumor comprising a first TIL population, depending on The condition is performed by surgical resection, biopsy biopsy, core biopsy, mini biopsy or other means used to obtain a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into multiple tumors fragments or tumor digests; (c) enzymatically digesting the plurality of tumor fragments to obtain the first TIL population; (d) optionally adding the tumor fragments or tumor digests to the closed system; (e ) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: Taserti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifoxin, Oridonin A, Gossin, Tenoride, Isolicorice Honokiol, lutein, and honokiol to generate a second population of TILs enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs, wherein the first amplification is optionally provided in the first Carried out in a closed container with a gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (d) to step (e) is optionally performed without opening the (f) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population , wherein the second amplification is performed for about 7-11 days to obtain the third TIL population, wherein the second amplification is optionally performed in a closed container providing a second gas-permeable surface area, and wherein from step (e) The transition to step (f) is optionally carried out without opening the system; (g) collecting the third TIL population obtained from step (f), wherein the transition from step (f) to step (g) is performed without opening the system as appropriate; (h) transferring the collected third TIL population from step (g) to an infusion bag, wherein the transfer from steps (g) to (h) depends on The case is carried out without opening the system; and (i) optionally genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time before the collecting step (g) , such that the third TIL population comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) resecting a tumor from a cancer in an individual or patient, the tumor comprising a first TIL population, depending on The condition is performed by surgical resection, biopsy biopsy, core biopsy, mini biopsy or other means used to obtain a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into multiple tumors fragments or tumor digests; (c) enzymatically digesting the plurality of tumor fragments to obtain the first TIL population; (d) optionally adding the tumor fragments or tumor digests to the closed system; (e ) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is optionally in an enclosure providing a first gas permeable surface area in a container, wherein the first amplification is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (e) to step (f) is optionally performed without opening the system (f) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cell (APC) and protein kinase B (AKT) inhibitors, optionally Wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonium Resin, Gossin, Tenolid, Isoliquiritigenin, Paretin, and Honokiol to generate a third TIL population that is CD39 ^LO /CD69 ^LO enriched and/or CD39/CD69 double negative TIL population , wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally prior to providing a second gas-permeable surface area carried out in a closed container, and wherein the transition from step (e) to step (f) is optionally carried out without opening the system; (g) collecting the third population of TILs obtained from step (f), wherein The transition from step (f) to step (g) is optionally performed without opening the system; (h) transferring the collected third TIL population from step (g) to an infusion bag, wherein The transfer of steps (g) to (h) is optionally performed without opening the system; and (i) optionally genetically modifying the first TIL population and/or the first TIL population at any time prior to the collecting step (g). The second population of TILs and/or the third population of TILs such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) resecting a tumor from a cancer in an individual or patient, the tumor comprising a first TIL population, depending on The condition is performed by surgical resection, biopsy biopsy, core biopsy, mini biopsy or other means used to obtain a sample from the cancer containing a mixture of tumor and TIL cells; (b) processing the tumor into multiple tumors fragments or tumor digests; (c) enzymatically digesting the plurality of tumor fragments to obtain the first TIL population; (d) optionally adding the tumor fragments or tumor digests to the closed system; (e ) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: Taserti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifoxin, Oridonin A, Gossin, Tenoride, Isolicorice Honokiol, lutein, and honokiol to generate a second population of TILs enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs, wherein the first amplification is optionally provided in the first Carried out in a closed container with a gas-permeable surface area, wherein the first amplification is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (d) to step (e) is optionally performed without opening the (f) by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, antigen presenting cells (APCs) and protein kinase B (AKT) inhibitors to perform a second TIL population amplified, optionally wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine , Rubescensin A, Gossin, Tenolide, Isoliquiritigenin, Parcetrin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 A double negative TIL population, wherein the second expansion is carried out for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is optionally provided in the second TIL population (2) carried out in a closed container with a gas-permeable surface area, and wherein the transition from step (e) to step (f) is carried out optionally without opening the system; Three TIL populations, wherein from step (f) to step ( The transformation of g) is carried out optionally without opening the system; (h) transferring the collected third TIL population from step (g) to an infusion bag, wherein steps (g) to (h) The transfer is optionally performed without opening the system; and (i) optionally genetically modifying the first TIL population and/or the second TIL population and/or the A third population of TILs such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and CD69. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: Means of obtaining a sample containing a mixture of tumor and TIL cells from a cancer in an individual or patient Obtaining and/or receiving a first TIL population; (b) selecting CD39 ^LO /CD69 ^LO from this first TIL population in (a) and/or CD39/CD69 double negative TILs to obtain a population of TILs enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative; (c) making the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double The negative TIL population is contacted with a first cell culture medium; (d) initial expansion of the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population in the first cell culture medium (or initiation of the first cell culture medium) amplified) to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody) and optionally antigen-presenting cells (APC), wherein the activated performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL -2. OKT-3 (anti-CD3 antibody) and APC; and wherein the rapid expansion is performed for a period of 14 days or less, optionally the rapid second amplification may be performed 1 after initiation of the rapid second amplification days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days; (f) collecting the third TIL population; and (g) optionally during the collecting step ( f) genetically modifying the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population and/or the second TIL population and/or the third TIL population at any time before such that the third TIL population Included are genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) by surgical excision, biopsy biopsy, core needle biopsy, mini-biopsy, or other Obtaining and/or receiving a first TIL population by means of obtaining a sample from a cancer in an individual or patient containing a mixture of tumor and TIL cells; CD3 antibody), optionally antigen presenting cells (APCs), and protein kinase B (AKT) inhibitors in cell culture medium for initial expansion (or initiation of the first expansion) by culturing the first TIL population, as appropriate Wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonium Resin, Gossythin, Tenolid, Isoliquiritigenin, Paretin, and Honokiol to generate a second TIL population that is CD39 ^LO /CD69 ^LO enriched and/or CD39/CD69 double negative TIL population , wherein the first amplification is optionally performed in a closed vessel providing a first gas-permeable surface area, wherein the initial first amplification is performed for about 1-8 days to obtain the second TIL population, and wherein from step ( a) the transition to step (b) is carried out optionally without opening the system; (c) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population ; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and APCs; and wherein the rapid expansion is performed for a period of 14 days or less, optionally the rapid second expansion can be at the rapid 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the second amplification; (d) collecting the third population of TILs; and ( e) optionally genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time prior to the collecting step (d) such that the third TIL population comprises genetically modified TILs, the genetically modified TILs comprise a genetic modification that reduces the expression of CD39 and CD69. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) by surgical excision, biopsy biopsy, core needle biopsy, mini-biopsy, or other means of obtaining a sample from a cancer in an individual or patient containing a mixture of tumor and TIL cells to obtain and/or receive a first TIL population; (b) performing an initial expansion of the first TIL population in a first cell culture medium ( or initiate the first expansion) to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (anti-CD3 antibody) and optionally antigen presenting cells (APC), wherein the initial first expansion is performed for a period of 1 to 8 days; (c) performing a rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT -3 (anti-CD3 antibody), APC and protein kinase B (AKT) inhibitors, optionally wherein the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068 , AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenolid, isoliquiritigenin, luteinin and honokiol to generate the third TIL population , which is a population of TILs enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative, wherein the rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion can be performed within the rapid second 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of the second amplification; (d) collecting the third TIL population; and (e ) optionally genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time prior to the collecting step (d) such that the third TIL population comprises genetically modified TILs , the genetically modified TILs comprise a genetic modification that reduces the expression of CD39 and CD69. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) by surgical excision, biopsy biopsy, core needle biopsy, mini-biopsy, or other Obtaining and/or receiving a first TIL population by means of obtaining a sample from a cancer in an individual or patient containing a mixture of tumor and TIL cells; CD3 antibody), optionally antigen presenting cells (APCs), and protein kinase B (AKT) inhibitors in cell culture medium for initial expansion (or initiation of the first expansion) by culturing the first TIL population, as appropriate Wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonium Resin, Gossythin, Tenolid, Isoliquiritigenin, Paretin, and Honokiol to generate a second TIL population that is CD39 ^LO /CD69 ^LO enriched and/or CD39/CD69 double negative TIL population , wherein the first amplification is optionally performed in a closed vessel providing a first gas-permeable surface area, wherein the initial first amplification is performed for about 1-8 days to obtain the second TIL population, and wherein from step ( a) the transition to step (b) is performed optionally without opening the system; (c) performing a rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium Comprising IL-2, OKT-3 (anti-CD3 antibody), APC and protein kinase B (AKT) inhibitors, optionally wherein the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183 , AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenolid, isoliquiritigenin, chrysanthemum and honokiol, To generate a third population of TILs that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion Amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after initiation of the rapid second amplification; (d) collecting the third and (e) optionally genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time prior to the collecting step (d), such that the third TIL population comprising genetically modified TILs comprising reduced CD39 and Genetic modification of CD69 expression. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the steps of: a) resecting a tumor from the individual or patient, the tumor comprising the first TIL population, depending on where by surgical resection, biopsy, core needle biopsy, mini biopsy or other means used to obtain a sample from the cancer containing a mixture of tumor and TIL cells; (b) fragmentation of the tumor into multiple tumors fragments or tumor digests; (c) select CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TILs from the first TIL population in such tumor fragments or tumor digests to obtain enriched CD39 ^LO /CD69 ^LO and and/or a CD39/CD69 double negative TIL population; (d) contacting the enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population with a first cell culture medium; (e) in the first cell culture medium The initial expansion (or initiation of the first expansion) of the CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population enriched in CD39 LO/CD69 LO and/or CD39/CD69 double negative TIL population is carried out in order to obtain the second TIL population, wherein the first cell culture medium comprises IL -2. Optional OKT-3 (anti-CD3 antibody) and optionally antigen-presenting cells (APC), wherein the initial first expansion is performed for a period of 1 to 8 days; (f) in the second cell performing a rapid second expansion of the second TIL population in culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and APC; and wherein the rapid expansion Performed for a period of 14 days or less, optionally the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days; (g) collecting the third TIL population; and (h) genetically modifying the enriched CD39 ^LO /CD69 ^LO and/or CD39/ CD69 double negative TIL population and/or the second TIL population and/or the third TIL population such that the third TIL population comprises genetically modified TILs comprising reduced expression of CD39 and CD69 of genetic modification. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the steps of: a) resecting a tumor from the individual or patient, the tumor comprising the first TIL population, depending on where by surgical resection, biopsy, core needle biopsy, mini biopsy or other means used to obtain a sample from the cancer containing a mixture of tumor and TIL cells; (b) fragmentation of the tumor into multiple tumors Fragments or tumor digests; (c) by combining IL-2, optionally OKT-3 (anti-CD3 antibody), optionally antigen presenting cells (APC) and protein kinase B (AKT) inhibitor Initial expansion (or initiation of first expansion) is performed by culturing the first TIL population in a first cell culture medium, optionally wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenoride, isoliquiritigenin, chrysanthemum and honokiol , to generate a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplification is optionally performed in a closed vessel providing a first gas-permeable surface area , wherein the initial first amplification is performed for about 1-8 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is optionally performed without opening the system; (d) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and APC and wherein the rapid amplification is performed for a period of 14 days or less, optionally the rapid second amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days after initiation of the rapid second amplification , 6 days, 7 days, 8 days, 9 days or 10 days; (e) collecting the third TIL population; and (f) optionally genetically modifying the first TIL population at any time prior to the collection (e) and /or the second population of TILs and/or the third population of TILs, such that the third population of TILs comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the steps of: a) resecting a tumor from the individual or patient, the tumor comprising the first TIL population, depending on where by surgical resection, biopsy, core needle biopsy, mini biopsy or other means used to obtain a sample from the cancer containing a mixture of tumor and TIL cells; (b) fragmentation of the tumor into multiple tumors fragments or tumor digests; (c) performing an initial expansion (or initiating a first expansion) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL -2. Optional OKT-3 (anti-CD3 antibody) and optionally antigen-presenting cells (APC), wherein the initial first expansion is performed for a period of 1 to 8 days; (d) in the second cell Rapid second expansion in culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), APC and protein kinase B (AKT) inhibitor, optionally wherein the The AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, Gossythin, tenolide, isoliquiritigenin, lutein, and honokiol to generate a third TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL population, wherein The rapid amplification is performed for a period of about 14 days or less, optionally the rapid second amplification can be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days after the initiation of the rapid second amplification. days, 7 days, 8 days, 9 days or 10 days; (e) collecting the third TIL population; and (f) optionally genetically modifying the first TIL population and/or at any time before the collection (e) The second population of TILs and/or the third population of TILs such that the third population of TILs comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising the steps of: a) resecting a tumor from the individual or patient, the tumor comprising the first TIL population, depending on where by surgical resection, biopsy, core needle biopsy, mini biopsy or other means used to obtain a sample from the cancer containing a mixture of tumor and TIL cells; (b) fragmentation of the tumor into multiple tumors Fragments or tumor digests; (c) by combining IL-2, optionally OKT-3 (anti-CD3 antibody), optionally antigen presenting cells (APC) and protein kinase B (AKT) inhibitor Initial expansion (or initiation of first expansion) is performed by culturing the first TIL population in a first cell culture medium, optionally wherein the AKT inhibitor is selected from the group consisting of palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenoride, isoliquiritigenin, chrysanthemum and honokiol , to generate a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the first amplification is optionally performed in a closed vessel providing a first gas-permeable surface area , wherein the initial first amplification is performed for about 1-8 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is optionally performed without opening the system; (d) Rapid second expansion in a second cell culture medium to obtain a third TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), APC and protein kinase B (AKT) Inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine , Rubescensin A, Gossin, Tenolide, Isoliquiritigenin, Parcetrin, and Honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 Double negative TIL population, wherein the rapid expansion is performed for a period of about 14 days or less, optionally the rapid second expansion can be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days; (e) collecting the third TIL population; and (f) genetically modifying the The first TIL population and/or the second TIL population and/or the third TIL population such that The third TIL population comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or dividing a tumor obtained from an individual into processing the sample into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual's cancer; (b) selecting CD39 ^LO /CD69 ^LO and and/or CD39/CD69 double-negative TILs to obtain enriched CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations; (c) by including IL-2, optionally OKT-3 and optionally Conditions comprising culturing the enriched CD39/CD69 double negative TIL population in cell culture medium of antigen presenting cells (APCs) for an initial first expansion to generate a second TIL population, wherein the initial first expansion is performed for about A first period of 1 to 11 days to obtain the second TIL population, wherein the number of the second TIL population is greater than the first TIL population; (d) by making the second TIL population with IL-2, OKT- 3 and APCs are contacted with a second cell culture medium to perform a rapid second expansion to produce a third TIL population, wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, wherein The third TIL population is a therapeutic TIL population; (e) collecting the therapeutic TIL population obtained from step (c); and (f) optionally genetically modifying the enrichment at any time prior to the collecting step (e) CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population and/or the second TIL population and/or the third TIL population, such that the third TIL population comprises genetically modified TILs, the genetically modified Modified TILs include genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or dividing a tumor obtained from an individual into The sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual's cancer; Initiating the first expansion by culturing the first TIL population in a first cell culture medium of a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693 , GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin A, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Magnolol, to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the initial first amplification is in a container comprising a first gas permeable surface area wherein the initial first amplification is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) by making The second TIL population is contacted with a second cell culture medium comprising IL-2, OKT-3, and APC to undergo a rapid second expansion to produce a third TIL population, wherein the rapid second expansion is performed for about 1 to 11 days (d) collecting the therapeutic TIL population obtained from step (c); and (e) optionally during the collection genetically modifying the first TIL population and/or the second TIL population and/or the third TIL population at any time prior to step (d) such that the third TIL population comprises genetically modified TILs, the genetically modified The TILs contain genetic modifications that reduce the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or dividing a tumor obtained from an individual into The sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual's cancer; (b) by including IL-2, optionally OKT-3, and optionally An initial first expansion is performed by culturing the first TIL population in cell culture medium of antigen presenting cells (APCs) to produce a second TIL population, wherein the initial first expansion is performed for a first period of about 1 to 11 days obtaining the second TIL population, wherein the number of the second TIL population is greater than the first TIL population; (c) by making the second TIL population and comprising IL-2, OKT-3, APC and protein kinase B ( Rapid secondary expansion by exposure to cell culture medium of an AKT inhibitor, where appropriate, wherein the AKT inhibitor is selected from the group consisting of: paltaxet, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930 , MK-2206, BAY 1125976, perifosine, oridonin, gossin, tenoride, isoliquiritigenin, luteinin and honokiol to produce the third TIL population, which is rich in collecting CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TIL populations, wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; (d) collecting the therapeutic TIL population obtained from step (c); and (e) optionally genetically modifying the first TIL population and/or the first TIL population at any time prior to the collecting step (d) The second population of TILs and/or the third population of TILs such that the third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces the expression of CD39 and CD69. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by processing a tumor sample obtained from a tumor into a plurality of tumor fragments or dividing a tumor obtained from an individual into The sample is processed into a tumor digest to obtain and/or receive a first TIL population derived from a tumor resected from the individual's cancer; Initiating the first expansion by culturing the first TIL population in a first cell culture medium of a protein kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: pataxerti, GSK690693 , GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin A, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Magnolol, to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the initial first amplification is in a container comprising a first gas permeable surface area wherein the initial first amplification is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) by making The second population of TILs is contacted with cell culture medium comprising IL-2, OKT-3, APC and a protein kinase B (AKT) inhibitor for rapid second expansion, optionally wherein the AKT inhibitor is selected from the group consisting of : Pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenolide, Isoliquiritigenin, luteinin, and honokiol to generate a third TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the rapid second amplification proceeds from about 1 to A second period of 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; (d) collecting the therapeutic TIL population obtained from step (c); and (e) optionally at At any time prior to the collecting step (d), the first TIL population and/or the second TIL population and/or the third TIL population are genetically modified such that the third TIL population comprises genetically modified TILs, which are Genetically modified TILs include genetic modifications that reduce the expression of CD39 and CD69. The method according to any one of claims 49 to 60, wherein in the initial first expansion step, the cell culture medium further comprises antigen-presenting cells (APCs), and wherein in the medium in the rapid second expansion step The number of APCs is greater than the number of APCs in the culture medium in the initial first expansion step. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) obtaining tumor-infiltrating lymphocytes (TILs) from a patient by surgical resection, biopsy, core needle biopsy, mini-biopsy, or other or a sample of cancer in an individual containing a mixture of tumor and TIL cells to obtain and/or receive a first TIL population, (b) select CD39 ^LO /CD69 ^LO and/or CD39/CD69 double-negative TILs to obtain enriched CD39 ^LO / CD69 ^LO and/or CD39/CD69 double-negative TIL populations; (c) by including IL-2, OKT-3 and antigen-presenting cells (APCs) culturing the TIL population enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative in the first cell culture medium to initiate the first expansion to produce a second TIL population, wherein the initial first expansion is In a vessel comprising a first gas-permeable surface area, wherein the initial first amplification is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first (d) optionally restimulating the second TIL population with OKT-3; (e) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises A genetic modification that reduces the expression of CD39 and CD69; (f) performing a rapid second expansion by culturing the modified second TIL population in a second medium comprising IL-2, OKT-3, and APC to produce A third TIL population, and wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain a therapeutic TIL population, wherein the third TIL population is a treatment comprising a genetic modification that reduces expression of CD39 and CD69 and (g) collecting the third TIL population. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) obtaining tumor-infiltrating lymphocytes (TILs) from a patient by surgical resection, biopsy, core needle biopsy, mini-biopsy, or other or cancer in an individual by means of obtaining and/or receiving a first TIL population from a sample containing a mixture of tumor and TIL cells, (b) by including IL-2, OKT-3, antigen presenting cells (APCs) and protein Initiating the first expansion by culturing the first TIL population in a first cell culture medium of a kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Hebu Parkol to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the initial first amplification is in a container comprising a first gas permeable surface area performing, wherein the initial first amplification is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) optionally with OKT -3 restimulating the second TIL population; (d) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces the expression of CD39 and CD69; (e) performing a rapid second expansion by culturing the modified second TIL population in a second medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the rapid second TIL population expanding for a second period of about 14 days or less to obtain the therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population comprising a genetic modification that reduces the expression of CD39 and CD69; and (f) collecting the Third TIL group. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) obtaining tumor-infiltrating lymphocytes (TILs) from a patient by surgical resection, biopsy, core needle biopsy, mini-biopsy, or other or cancer in an individual by means of obtaining and/or receiving a first TIL population from a sample containing a mixture of tumor and TIL cells, (b) by presenting in a first TIL population comprising IL-2, OKT-3 and antigen presenting cells (APCs) The initial first expansion is performed by culturing the first TIL population in a two-cell culture medium to produce a second TIL population, wherein the initial first expansion is performed in a container comprising a first gas permeable surface area, wherein the initial first Expansion is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) optionally restimulating the second TIL population with OKT-3 population; (d) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces the expression of CD39 and CD69; (e) by including IL -2, OKT-3, APC and the second culture medium of protein kinase B (AKT) inhibitor cultivate this modified second TIL population to carry out rapid second expansion, wherein the AKT inhibitor is selected from the following as appropriate Constituent group: pataseti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, cotton xanthin, tenol Lido, isoliquiritigenin, chrysanthemum, and honokiol to generate a third TIL population enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the rapid second amplification was performed A second period of about 14 days or less to obtain the therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population comprising a genetic modification that reduces the expression of CD39 and CD69; and (f) collecting the third TIL population group. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) obtaining tumor-infiltrating lymphocytes (TILs) from a patient by surgical resection, biopsy, core needle biopsy, mini-biopsy, or other or cancer in an individual by means of obtaining and/or receiving a first TIL population from a sample containing a mixture of tumor and TIL cells, (b) by including IL-2, OKT-3, antigen presenting cells (APCs) and protein Initiating the first expansion by culturing the first TIL population in a first cell culture medium of a kinase B (AKT) inhibitor, optionally wherein the AKT inhibitor is selected from the group consisting of: palaxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosin, Oridonin, Gossin, Tenoride, Isoliquiritigenin, Huangpi and Hebu Parkol to produce a second TIL population that is enriched for CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL populations, wherein the initial first amplification is in a container comprising a first gas permeable surface area performing, wherein the initial first amplification is performed for a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) optionally with OKT -3 restimulating the second TIL population; (d) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces the expression of CD39 and CD69; (e) rapid second expansion by culturing the modified second TIL population in a second medium comprising IL-2, OKT-3, APC, and protein kinase B (AKT) inhibitors, optionally wherein The AKT inhibitor is selected from the group consisting of pataxerti, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin , gossinthin, tenolide, isoliquiritigenin, chrysanthemum and honokiol to generate a third TIL population, which is enriched in CD39 ^LO /CD69 ^LO and/or CD39/CD69 double negative TIL population, wherein the rapid second expansion is performed for a second period of about 14 days or less to obtain the therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population comprising a genetic modification that reduces expression of CD39 and CD69; and (f) Collect the third TIL population. The method according to any one of claims 1 to 67, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, three Negative breast cancer, cancers caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer and renal cell carcinoma. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprising: (a) by adding IL-2, optionally OKT-3, and optionally antigen presenting cells Initiating the first expansion by culturing a TIL population enriched in CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO in a cell culture medium of (APC) to generate a second population of TILs, wherein the initiating first expansion performing a first period of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (b) by combining the second TIL population with IL-2, Contacting a second cell culture medium of OKT-3 and APCs for a rapid second expansion to produce a third TIL population, wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population , wherein the third TIL population is a therapeutic TIL population; and (c) collecting the third TIL population obtained from step (b); (d) genetically modifying the enrichment at any time prior to the collecting step (c) CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO TIL population, the second TIL population and/or the third TIL population, such that the collected third TIL population comprises genetically modified TILs that were Genetically modified TILs include genetic modifications that reduce the expression of CD39 and CD69. The method of claim 67, wherein in step (a), the cell culture medium further comprises antigen-presenting cells (APCs), and wherein the number of APCs in the culture medium in step (b) is greater than the number of APCs in step (b). Number of APCs in culture medium. A method for expanding T cells, comprising: (a) priming a first TIL population obtained from a donor by culturing the first TIL population to effect growth and initiate activation of the first T cell population Initiating a first amplification, wherein the first TIL population is a TIL population enriched for CD39/CD69 double negative and/or CD39 ^LO /CD69 ^LO ; (b) of the first TIL population initiated in step (a) After activation begins to decay, a rapid second expansion of the first TIL population is performed by culturing the first TIL population to achieve growth and enhance activation of the first T cell population to obtain a second T cell population; (c ) collecting the second T-cell population; and (d) genetically modifying the first TIL population and/or the second TIL population such that the collected second TIL population comprises genetically modified TILs that are genetically modified The TILs contain genetic modifications that reduce the expression of CD39 and CD69. A method for expanding T cells comprising: (a) performing a first T cell population from a tumor sample by culturing the first T cell population to achieve growth and initiate activation of the first T cell population Initiating first expansion of a tumor sample obtained from one or more mini-biopsy, core-needle biopsy, or needle biopsy of a tumor in a donor, wherein the first T cell population is enriched for CD39/CD69 double negative and/or a T cell population of CD39 ^LO /CD69 ^LO ; (b) after the activation of the first T cell population initiated in step (a) has started to decay, by culturing the first T cell population to achieve growth and enhancing activation of the first T cell population to perform a rapid second expansion of the first T cell population to obtain a second T cell population; and (c) collecting the second T cell population; and (d) genetic modification The first T cell population and/or the second TIL population such that the collected second T cell population comprises genetically modified TILs comprising genetic modifications that reduce the expression of CD39 and CD69. The method of any one of claims 1 to 12, 29 to 45, or 57 to 60, wherein the second TIL population from the first expansion or the third TIL population from the second expansion or both Make this modification. The method of any one of claims 13 to 20, 25 to 28, 46 to 56, and 62 to 67, wherein the second TIL population from the initial first expansion or the second TIL population from the rapid second expansion Three TIL populations or both carry out this modification. The method of any one of claims 1 to 12, 29 to 45 or 57 to 60, wherein the modification is performed on the second population of TILs from the first amplification and before the second amplification. The method of any one of claims 13 to 20, 25 to 28, 46 to 56, and 62 to 67, wherein for the second TIL population from the initial first expansion and before the rapid second expansion or This modification was carried out in both cases. The method of any one of claims 1 to 12, 29 to 45 or 57 to 60, wherein the modification is performed on a third population of TILs from the second amplification. The method of any one of claims 13 to 20, 25 to 28, 46 to 56 and 62 to 67, wherein the modification is performed on a third population of TILs from the rapid second amplification. The method according to any one of claims 1 to 20, 25 to 60 and 62 to 69, wherein the modification is performed after the collecting. The method of any one of claims 1-12, 29-45, or 57-60, wherein the first amplification is performed for a period of about 11 days. The method of any one of claims 13-28 or 49-69, wherein the initial first amplification is performed for a period of about 11 days. The method of any one of claims 1 to 12, 29 to 45, or 57 to 60, wherein the IL-2 is present in the first amplification at an initial concentration between 1000 IU/mL and 6000 IU/mL in the cell culture medium. The method of any one of claims 5 to 8 or 14 to 22, wherein the IL-2 is present in the cell culture medium in the initial first expansion at a concentration between 1000 IU/mL and 6000 IU/mL . The method according to any one of claims 1 to 12, 29 to 45 or 57 to 60, wherein in the second amplification step, the IL-2 is at an initial concentration between 1000 IU/mL and 6000 IU/mL present and the OKT-3 antibody was present at an initial concentration of approximately 30 ng/mL. The method of any one of claims 13 to 28 or 49 to 69, wherein in the rapid second amplification step, the IL-2 is present at an initial concentration between 1000 IU/mL and 6000 IU/mL and the OKT-3 antibody was present at an initial concentration of approximately 30 ng/mL. The method of claims 1 to 12, 29 to 45 or 57 to 60, wherein a gas-permeable container is used for the first amplification. The method according to any one of claims 13 to 28 or 49 to 69, wherein the initial first amplification is performed using a gas permeable container. The method according to any one of claims 1 to 12, 29 to 45 or 57 to 60, wherein a gas permeable container is used for the second amplification. The method of claims 13 to 28 or 49 to 69, wherein a gas-permeable container is used for the rapid second amplification. The method according to any one of claims 1 to 12, 29 to 45 or 57 to 60, wherein the first expanded cell culture medium further comprises a cytokine selected from the group consisting of: IL-4, IL-7 , IL-15, IL-21 and combinations thereof. The method of claims 13 to 28 or 49 to 69, wherein the cell culture medium for initiating the first expansion further comprises a cytokine selected from the group consisting of: IL-4, IL-7, IL-15, IL -21 and combinations thereof. The method according to any one of claims 1 to 12, 29 to 45 or 57 to 60, wherein the second expanded cell culture medium further comprises a cytokine selected from the group consisting of: IL-4, IL-7 , IL-15, IL-21 and combinations thereof. The method according to any one of claims 13 to 28 or 49 to 69, wherein the cell culture medium of the rapid second expansion further comprises a cytokine selected from the group consisting of: IL-4, IL-7, IL- 15. IL-21 and combinations thereof. The method of any one of claims 1 to 24, further comprising the step of treating the patient with non-myeloablative lymphodepleting therapy prior to administering the third TIL population to the patient. The method of claim 92, wherein the nonmyeloablative lymphocyte depletion therapy comprises administering cyclophosphamide at a dose of 60 mg/m ² /day for two days, followed by administration of 25 mg/m ² /day Step with fludarabine for three consecutive days. The method of claim 92, wherein the nonmyeloablative lymphocyte depletion therapy comprises administering cyclophosphamide at a dose of 60 mg/m ² /day and fludala at a dose of 25 mg/m ² /day Fludarabine was administered for two days, followed by a three-day step of administering fludarabine at a dose of 25 mg/m ² /day. The method of claim 92, wherein the nonmyeloablative lymphocyte depletion therapy comprises administering cyclophosphamide at a dose of 60 mg/m ² /day and fludala at a dose of 25 mg/m ² /day Fludarabine was administered for two days, followed by a one-day step of administering fludarabine at a dose of 25 mg/m ² /day. The method of any one of claims 93 to 95, wherein the cyclophosphamide is administered together with mesna. The method of any one of claims 1 to 24 or 92 to 96, further comprising the step of starting to treat the patient with an IL-2 regimen the day after administering TIL to the patient. The method of any one of claims 1 to 24 or 92 to 96, further comprising the step of initiating treatment of the patient with an IL-2 regimen on the same day as TIL is administered to the patient. The method of claim 97 or 98, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg of aldesleukin or a biosimilar or variant thereof, which is Administer as a 15-minute bolus intravenous infusion every eight hours until tolerated. The method of any one of claims 1 to 24 or 92 to 96, wherein a therapeutically effective TIL population is administered and the TIL population comprises about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ TILs. The method of any one of claims 13-28 or 49-69, wherein the initial first amplification and rapid second amplification are performed for a period of 21 days or less. The method of any one of claims 13 to 28 or 49 to 69, wherein the initial first amplification and rapid second amplification are performed for a period of 16 or 17 days or less. The method of any one of claims 13 to 28 or 49 to 69, wherein the initial first amplification is performed for a period of 7 or 8 days or less. The method of any one of claims 13-28 or 49-69, wherein the rapid second amplification is performed for a period of 11 days or less. The method according to any one of claims 1 to 12, 29 to 45 or 57 to 60, the first amplification and the second amplification are each individually performed within a period of 11 days. The method according to any one of claims 21 to 24 or 61, wherein steps (a) to (f) are performed within about 26 days. The method of any one of claims 1 to 106, wherein the genetically modified TILs further comprise additional genetic modifications that reduce the expression of one or more immune checkpoint genes selected from the group comprising : CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3 and TOX. The method of claim 107, wherein the one or more immune checkpoint genes are selected from the group comprising: PD-1, CBL-B, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TIGIT , TET2, TGFβ and PKA. The method of any one of claims 1 to 108, wherein the genetically modified TILs further comprise additional genetic modifications that induce one or more immune checkpoint genes in at least a portion of the therapeutic TIL population Enhanced expression of immune checkpoint genes selected from the group comprising: CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL -21. NOTCH 1/2 intracellular domain (ICD) and/or NOTCH ligand mDLL1. The method of any one of claims 1 to 109, wherein the genetic modification step is performed using a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks at the one or more immune checkpoint genes. The method according to any one of claims 1 to 110, wherein the genetic modification is performed using one or more methods selected from the group consisting of: CRISPR method, TALE method, zinc finger method and combinations thereof. The method of claim 111, wherein the methods comprise CRISPR methods. The method of claim 112, wherein the CRISPR method is a CRISPR/Cas9 method. The method according to claim 111, wherein the genetic modification comprises TALE method. The method according to claim 111, wherein the genetic modification comprises zinc finger method. The method of any one of claims 1 to 115, wherein processing the tumor sample obtained from the individual into a tumor digest comprises culturing the tumor sample in an enzyme medium. The method of any one of claims 1 to 115, wherein processing the tumor sample obtained from the individual into a tumor digest further comprises mechanically disrupting the tumor sample to dissociate the tumor sample. The method of any one of claims 1 to 115, wherein processing the tumor sample obtained from the individual into a tumor digest further comprises purifying the dissociated tumor sample using density gradient separation. The method according to claim 116, wherein the enzyme medium comprises DNase. The method according to claim 116, wherein the enzyme medium contains 30 units/ml of DNase. The method of claim 116, wherein the enzyme medium comprises collagenase. The method of claim 116, wherein the enzyme culture medium contains 1.0 mg/mL of collagenase. The method of any one of claims 1 to 122, wherein the collected population of therapeutic TILs comprises sufficient TILs to administer a therapeutically effective dose to the individual. The method of any one of claims 1 to 123, wherein the therapeutically effective dose comprises about 1×10 ⁹ to about 9×10 ¹⁰ TILs. The method of any one of claims 1 to 124, wherein the APCs comprise peripheral blood mononuclear cells (PBMCs). The method of any one of claims 1 to 125, wherein the therapeutic TIL population collected in step (e) exhibits an increased CD8+ cell subset relative to the first and/or second TIL population. 11. The method of any one of claims 1 to 126, wherein the PBMCs are supplemented at a ratio of TIL:PBMCs of about 1:25. The method of any one of claims 1 to 127, wherein the first amplification in step and the second amplification in step are each individually performed within a period of 11 to 12 days. The method of any one of claims 1 to 128, wherein steps (a) to (e), (f) or (g) are performed within about 10 days to about 24 days. The method of any one of claims 1 to 129, wherein steps (a) to (e), (f) or (g) are performed within about 15 days to about 24 days. The method according to any one of claims 1 to 130, wherein steps (a) to (e), (f) or (g) are performed within about 20 days to about 24 days. The method according to any one of claims 1 to 131, wherein steps (a) to (e), (f) or (g) are performed within about 20 days to about 22 days. The method of any one of claims 1 to 132, wherein the second population of TILs is at least 50 times greater in number than the first population of TILs. A TIL population according to any one of the methods of claims 1-133. A composition comprising a population of TILs according to any one of the methods of claims 1-134.

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